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

The Yale University Institutional Animal Care and Use Committee approve all the in vivo work described. Sample collection was performed with patient consent and approved by the Human Investigations Committee of Yale University School of Medicine.

1. Preparation of Human Ovarian Cancer Cells

1. Before preparing the cells make sure all materials required for the intra-uterine injection described below are ready and easily accessible. Inject the cell suspension as soon as it is ready.
   1. Grow fluorescent-labeled cancer cells in culture. We use an F2-generation of human CD44+/MyD88+ epithelial ovarian cancer cells, which we previously demonstrated to harbor stemness properties such as tumorigenicity, chemoresistance and high capacity for tumor repair ^14-19. These cells were generated to stably express mCherry fluorescence (F2-mCherry, Fig. 1) using lentivirus produced from the polyethylenimine (PEI) protocol ^20,21.
   2. On day of injection, harvest the cells by trypsinization and wash once with phosphate buffered saline (PBS).
   3. Determine cell count and suspend the cells at 3 x 10⁶ cells/ 50 µl in appropriate growth media. For the ovarian cancer cells we use RPMI 160 with 10% FBS. Keep the cells in the incubator until ready to inject but do not keep longer than 15 min. This is enough for one mouse injection.

2. Intra-uterine Injection of Human Ovarian Cancer Cells in Athymic Nude Mice

Note that the following procedure requires assistance from a second person. In addition, since this is survival surgery, use sterile surgical instruments. This survival surgery procedure should take approximately 10 to 15 min.

1. Anesthetize the mouse using 2% isoflurane. 7- to 8-week-old athymic nude mice is typically used.
2. Verify that the animal is completely anesthesized by pinching the foot pad using fingers or forceps. The animal should be completely non-responsive to the pain prior to starting the surgery.
3. Place the anesthetized mouse ventral side up on a sterile gauze pad with the head away from the investigator.
4. Apply ointment on eyes with a cotton-tip applicator to prevent dryness while under anesthesia.
5. Immediately insert the head in a nose cone system connected to an isoflurane vaporizer (Fig. 2A). This delivers the anesthesia throughout the surgical procedure.
6. Disinfect the left (or right) side of the abdomen using alcohol pads (Fig. 2B) followed by iodine then place a sterile surgical drape over the surgical site.
7. Using sterile surgical scissors and forceps make a 1-2 cm skin incision on the lower left quadrant of the mouse (Fig. 2C).
8. Lift the muscle and make an incision in order to reach the peritoneum.
9. Dissect the oblique muscle to expose the abdominal cavity.
10. Locate and identify the left uterine horn (Fig. 2D).
11. Using sterile hemostat, clamp both anterior and posterior sides of the horn (Fig. 2E). Place the anterior clamp right below the fallopian tube and the posterior clamp right above the cervix.
12. Have the second person inject the cell suspension. Place the needle containing cells, at 45° angle perpendicular to the horn (Fig. 2F). Very slowly inject 50 µl of cell suspension into the lumen of the uterine horn.
13. Remove the needle and release the anterior clamp.
14. Release the posterior clamp and replace the uterine horn in the abdominal cavity.
15. Close the peritoneum using a synthetic absorbable suture and close the skin using a tissue adhesive.
16. Remove the mouse from the nose cone and place back in the cage. If necessary, change the beddings to ensure a clean cage after the surgery. Make sure all animals are awake and active before leaving unattended.
17. Provide Ibuprofen at 0.11 mg/ml of drinking water for the first 48 hr post surgery. Afterwards, replace with regular drinking water. 
    1. Note: that if athymic nude mice are used and the skin is closed using tissue adhesive instead of sutures, it is not necessary to separate the mice after surgery. SCID mice are more aggressive and may have to be separated.
18. Closely monitor the site of incision for possible infection due to open wound.

3. Detection of Early Stage Intra-peritoneal Ovarian Cancer by Live In Vivo Imaging

Determine the presence of intra-peritoneal i.p disease by live in vivo imaging. A Multimodal Animal Rotation System (MARS) is used in this protocol.

1. Access the MARS capture software from within the MI software. This module provides coordinated control of the MARS capture settings while leveraging the imaging system’s capture and analysis capabilities. Optimize individual capture settings for each modality using a single-view imaging prior to developing the rotation protocol in step 3.4.
2. Input predetermined capture values. For fluorescence capture, use 10 sec exposure, 2 x 2 binning, f-stop 2.8, FOV 120 mm, ex: 550 nm, em: 600 nm filter capture setting. For X-ray capture, use a 10 sec exposure, 2 x 2 binning, f-stop 2.8, FOV 120 mm, 35 kVp, 0.4 mm Al filter capture setting.
3. Save the parameters in step 3.2 as individual session files.
4. Create a rotation sequence protocol by selecting the Create/Edit Protocols button from the system interface acquire window.
5. Select the previously saved fluorescence session file and save as Step One. Next, select the corresponding X-ray session file and save as Step Two.
6. 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 for seamless visualization of features. The specific values include a starting angle of -180 degrees, a range of 375 degrees, and an increment of 15 degrees.
7. Save the protocol and click the Done button to initialize the MARS and map the stepper motor positions corresponding to each of the angle increments.
8. Perform the final calibration as described below to align the ventral, dorsal and lateral views for the specific animal positioned in the animal support with specific degrees of rotation to be used by the software protocol.
9. Anesthetize the mouse using a mixture of medical air and 2% isoflurane at a flow rate of 2 L/min.
10. Start the alignment by selecting the Preview button from within the Acquire menu to bring up the Rotator tab. Use the reflective light modality rather than the multiwavelength or X-ray modalities to expedite the process.
11. Select the Load Mouse option and proceed through the series of positioning menus.
12. Ensure that the tubed end of the collapsible nosecone is in the nosecone recess and place the mouse in the prone orientation with its head in the nosecone.
13. Begin the calibration at the 0 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.
14. Repeat the process as prompted for -180, – 90, +90, and +180 degree positions and click Done.
15. Execute the previously saved protocol by clicking the Execute Protocol button from the main acquire window. During the execution of the protocol, observe a status window providing real time status updates for the Current Capture, Exposure Duration, and overall Protocol step.
16. Assemble individual images into a movie format using the Rotation software. Use the Display Controls to adjust brightness/contrast, transparency, and to set the display color for the fluorescence signal.
17. Export the final sequence of images in the *.AVI file format for presentation.

4. Administration of 1^st and 2^nd Round Chemotherapy

1. Following i.p. tumor detection by MARS, determine the initial tumor burden region of interest (ROI) using standard two-dimensional positioning. This is performed on a transparent animal tray fitted in the imaging system as described below in Section 5. Once initial ROI is determined, administer 4 doses of 12 mg/kg Paclitaxel i.p. every three days. Paclitaxel is obtained from Hospira Inc. in a ready-to-go formulation with cremaphor as vehicle. We previously demonstrated that this treatment regimen has significant anti-tumor activity against the xenograft model compared to vehicle control and induces complete eradication of detectable tumor burden ²⁰. However, due to the nature of the disease, i.p. tumors recur a few days after treatment ends ²⁰.
2. Monitor response to treatment by imaging every 3 days using standard two-dimensional positioning.
3. After the fourth dose of Paclitaxel, classify the mice as having complete response (ROI less than 2,000), partial response (up to 35% decrease from the initial ROI obtained from step 4.1, stable disease (up to 50% increase from the initial ROI), or progression with treatment (more than 50% increase from initial ROI) based on the fluorescent signal observed.
4. For mice with complete response, monitor recurrence by imaging every 3 days. Once recurrence is observed (ROI = to initial ROI in step 4.1), re-initiate the second round of Paclitaxel as described in 4.1.
5. In mice with partial disease, stable disease, or those that progressed with treatment, re-initiate second round of Paclitaxel 3 days after the 4^th dose of Paclitaxel. Similarly, image these every three days.

5. Monitoring Response to Treatment Using Standard Two-dimensional Positioning

1. Anesthetize the mice to be imaged using a mixture of medical air and 2% isoflurane at a flow rate of 2 L/min.
2. Place the head of the anesthetized mice in individual nosecones in the transparent animal tray within the imaging chamber (Fig. 3).
3. Input the predetermined exposure parameters for fluorescence (10 sec exposure, 2 x 2 binning, f-stop 2.8, FOV 120 mm, ex: 550 nm, em: 600 nm) and X-ray (20 sec exposure, 2 x 2 binning, f-stop 2.8, FOV 120 mm, 35 kVp, 0.4 mm Al filter).
4. Click Expose to capture both images in sequence.
5. Open the images in the Bruker MI software for visual inspection and region of interest analysis.

6. Image Overlay and Analysis

To provide a valid comparison for both visual and quantitative analysis of the time course image sequence process all foreground and background images similarly.

1. Open both the foreground image (i.e. fluorescence) and the background imaging in the software and select the tile function from the Window menu at the top (Fig. 4A).
2. To process the foreground image; open the Image Display tab on the top bar (Fig. 4A, red arrow) and set the min/max values to 260 and 5,000, respectively (Fig. 4A, blue arrow)
   Note: Image Display range differs from study to study depending upon the fluorescence signal intensities captured in images. Images should be contrasted with a min/max that will show tumor masses, yet not show background from autofluorescence of the animal.
3. Next open Auto-ROIs Tab from the Navigation Panel on the left (Fig. 4B, red arrow) and select the New ROI Set from the menu (Fig. 4C, red arrow). Adjust the settings to use the Threshold algorithm at 630 grayscale levels and deselect the Restrict size Max to ensure all potential ROI’s are recorded (Fig. 4C).
   Note: Threshold settings may differ from study to study depending upon the fluorescence signal intensities captured in images. Images thresholded to quantitate signal from tumor masses yet not measure signal from background from autofluorescence of the animal.
4. From within the Image Display Window select the Mask function. Now only the area corresponding to the pixels that have values equal to or exceeding the threshold value of 630 grayscale levels will be displayed.
5. To view the quantitative results select the Analysis tab from the top menu bar and the select the display tab (Fig. 4D, red arrows).
6. From the Display menu select the Serial Number, Sum, Mean, and Area check boxes and then click OK.
7. Save the image so the values can be exported along once all fluorescence images are processed.
8. Next, highlight the X-ray image and again open the Image Display from the top menu bar. Set the values for min, max and gamma to give the best visual representation of the mouse skeleton.
   Note: Since no quantitation is performed on the X-ray images each individual X-ray image from the time sequence can be processed (i.e. min, max and gamma values) to produce the best looking representation of that data set.
9. Again with the X-ray image highlighted select the Overlay check box and choose the corresponding fluorescence image file name from the drop down menu (Fig. 4E).
10. To extract the overlayed output, select the Annotations tab from the Navigation menu (Fig. 4B, blue arrow) on the left. Select appropriate tabs to add any additional data or text and finally add the intensity scale bar so the fluorescence intensity data can be quickly determined from the image.
11. Once the annotations are completed highlight all the objects on the Annotations page and select copy from the Edit menu at the top and paste the overlayed image data into the appropriate file (e.g. Word, PowerPoint, and/or Photoshop) for display.
12. Repeat Steps 6.1- 6.11 for all pairs of fluorescence and X-ray images in the sequence. Be sure to safe all images before closing them.
13. For final quantitation open all the fluorescence images at the same time and select Tile from the Window menu along the top.
14. Next select the Export All Open Documents to view the data.
    Note: In absence of Excel program, deselect the tab. MI will generate a tab delimited text (i.e. .txt) file that can be transferred to an appropriate computer for further analysis.