Name: murine model for non-invasive imaging to detect and monitor ovarian cancer recurrence
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Description: Watch this Scientific Journal Video about Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence at JoVE.com

This study was supported by NIH grants RO1CA118678 and RO1CA127913, by the Sands Family Foundation, and the Discovery to Cure Program.

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
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>On day of injection, harvest the cells by trypsinization and wash once with phosphate buffered saline (PBS).</li>
		<li>Determine cell count and suspend the cells at 3 x 106 cells/ 50 &#181;l 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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Anesthetize the mouse using 2% isoflurane. 7- to 8-week-old athymic nude mice is typically used.</li>
	<li>Verify that the animal is completely anesthesized by pinching the foot pad using fingers or forceps.&#160;The animal should be completely non-responsive to the pain prior to starting the surgery.</li>
	<li>Place the anesthetized mouse ventral side up on a sterile gauze pad with the head away from the investigator.</li>
	<li>Apply ointment on eyes with a cotton-tip applicator to prevent dryness while under anesthesia.</li>
	<li>Immediately insert the head in a nose cone system connected to an isoflurane vaporizer (Fig. 2A). This delivers the anesthesia throughout the surgical procedure.</li>
	<li>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.</li>
	<li>Using sterile surgical scissors and forceps make a 1-2 cm skin incision on the lower left quadrant of the mouse (Fig. 2C).</li>
	<li>Lift the muscle and make an incision in order to reach the peritoneum.</li>
	<li>Dissect the oblique muscle to expose the abdominal cavity.</li>
	<li>Locate and identify the left uterine horn (Fig. 2D).</li>
	<li>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.</li>
	<li>Have the second person inject the cell suspension. Place the needle containing cells, at 45&#176; angle perpendicular to the horn (Fig. 2F). Very slowly inject 50 &#181;l of cell suspension into the lumen of the uterine horn.</li>
	<li>Remove the needle and release the anterior clamp.</li>
	<li>Release the posterior clamp and replace the uterine horn in the abdominal cavity.</li>
	<li>Close the peritoneum using a synthetic absorbable suture and close the skin using a tissue adhesive.</li>
	<li>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.&#160;Make sure all animals are awake and active before leaving unattended.</li>
	<li>Provide Ibuprofen at 0.11 mg/ml of drinking water for the first 48 hr post surgery.&#160;Afterwards, replace with regular drinking water.&#160;
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Closely monitor the site of incision for possible infection due to open wound.</li>
</ol>
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.
<ol>
	<li>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&#8217;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.</li>
	<li>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.</li>
	<li>Save the parameters in step 3.2 as individual session files.</li>
	<li>Create a rotation sequence protocol by selecting the Create/Edit Protocols button from the system interface acquire window.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Anesthetize the mouse using a mixture of medical air and 2% isoflurane at a flow rate of 2 L/min.</li>
	<li>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.</li>
	<li>Select the Load Mouse option and proceed through the series of positioning menus.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat the process as prompted for -180, - 90, +90, and +180 degree positions and click Done.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Export the final sequence of images in the *.AVI file format for presentation.</li>
</ol>
4. Administration of 1st and 2nd Round Chemotherapy
<ol>
	<li>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.</li>
	<li>Monitor response to treatment by imaging every 3 days using standard two-dimensional positioning.</li>
	<li>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.</li>
	<li>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.</li>
	<li>In mice with partial disease, stable disease, or those that progressed with treatment, re-initiate second round of Paclitaxel 3 days after the 4th dose of Paclitaxel. Similarly, image these every three days.</li>
</ol>
5. Monitoring Response to Treatment Using Standard Two-dimensional Positioning
<ol>
	<li>Anesthetize the mice to be imaged using a mixture of medical air and 2% isoflurane at a flow rate of 2 L/min.</li>
	<li>Place the head of the anesthetized mice in individual nosecones in the transparent animal tray within the imaging chamber (Fig. 3).</li>
	<li>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).</li>
	<li>Click Expose to capture both images in sequence.</li>
	<li>Open the images in the Bruker MI software for visual inspection and region of interest analysis.</li>
</ol>
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.
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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&#8217;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.</li>
	<li>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.</li>
	<li>To view the quantitative results select the Analysis tab from the top menu bar and the select the display tab (Fig. 4D, red arrows).</li>
	<li>From the Display menu select the Serial Number, Sum, Mean, and Area check boxes and then click OK.</li>
	<li>Save the image so the values can be exported along once all fluorescence images are processed.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>For final quantitation open all the fluorescence images at the same time and select Tile from the Window menu along the top.</li>
	<li>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.</li>
</ol>

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Publication fees for this article were partially sponsored by Bruker Corporation.

We describe a protocol to establish an i.p. human ovarian cancer animal model that mimics the clinical profile observed in patients. In addition, we describe the use of an animal rotation device that addresses the sensitivity limitation of 2D imaging. Taken together, these techniques can serve as platforms to discover novel compounds that can target chemoresistant recurrent ovarian cancer. In addition, such model can be used to understand the biology of cancer recurrence and progression.
Due to its retroperitoneal location, early-stage i.p. ovarian cancer xenografts are almost impossible to detect by physically examining the mouse. In most cases, once the disease can be palpated, the tumor burden is already significant and therefore limits the evaluation of treatment efficacy. The use of fluorescently labeled cells allows us to assess the establishment of i.p. tumor in real time and consequently identifying the optimum time to begin treatment. In a similar way, fluorescent-labeled xenografts allow monitoring of treatment response. It should be pointed-out however that i.p. tumors deeper than 1 cm are typically not detectable irrespective of the reporter system.
The use of human ovarian cancer stem cells 14,15,17,22 generates xenografts that mimic the clinical profile observed in patients. As a primary disease, the model is responsive to Paclitaxel but cessation of treatment eventually leads to chemoresistant recurrent disease. Introducing the cells through the uterine horns at the density specified in the Protocol section usually results in ovarian tumors within 10 days with a few peritoneal implants, and therefore mimics early-stage disease. The use of fluorescently labeled cells allows us to assess the establishment of i.p. tumor in real- time and consequently identifying the optimum time to begin treatment. In a similar way, fluorescent-labeled xenografts allow monitoring of response to treatment. If other types of cancer cell lines are used, ovarian or otherwise, it is possible that this profile may not be observed. When SKOV3 is used for example, it has been reported that the initial i.p. tumors are already resistant 23. Nevertheless, if labeled with a reporter such as fluorescence, i.p, disease can be followed in real-time.
If other fluorescent reporter is used, it is important to perform initial imaging with a control (no tumor) animal. This will allow optimization of imaging protocol to achieve the best background to signal ratio. In our experience, nude mice typically have high background when imaged using the GFP acquisition settings.
It is important that cells injected intra-uterine are in single suspension to avoid the establishment of tumors in the uterus. It is also important to avoid scratching the uterine epithelial layer, which also facilitates engraftment of the cancer cells in the uterus thus producing an intra-uterine tumor instead of an i.p. disease. In addition, during data analysis, it is important to set the gamma value to 1. This insures that the intensity of the images is linear and allows comparison between images.
During the acquisition of MARS images, it is important to ensure that the tubed end of the collapsible nosecone is in the nosecone recess. The nosecone serves as a point of contact for the mouse and is therefore required for obtaining precisely calibrated angles. For longer imaging protocols (i.e. longer than 1 hour), inject 100 &#956;l of sterile saline subcutaneously to help prevent dehydration. Animal body temperature should be maintained using warm air flowed through the system at approximately 37&#176;C. A limitation of the MARS system is that only one animal can be imaged at a time with a total run time of about 1 hour per animal.
In conclusion, we describe the establishment of an animal model that closely mimics ovarian cancer, both primary and recurrent disease. This model can be used to evaluate the efficacy of novel diagnostic or therapeutic modalities.

Animal models are indispensible tools in life science research. In cancer particularly, data acquired from animal studies provide the necessary information required to initiate the testing of novel diagnostic or therapeutic applications in humans 1-3. Animal models for solid cancers are classically established subcutaneously as it provides an easy means to measure tumor burden and evaluate treatment efficacy without having to sacrifice the animals. Indeed, intra-peritoneal (i.p.) models require that animals be sacrificed to detect and measure any changes in tumor growth. However, for i.p. cancers such ovarian cancer, orthotropic models offer the advantage of studying the disease in its proper environment4-6. For such a model to be of use in the evaluation of anti-tumor activity, non-invasive imaging methods need to be developed that allow quantification of i.p. tumor burden in live mice.
A major challenge in the use of i.p. animal models is the difficulty in accurately quantifying tumor burden by physical examination. Accurate quantification of i.p. tumors usually require the mice to be sacrificed for dissection. This approach requires the use of high number of animals, which would be sacrificed at different time points. In addition to the cost, it introduces high data variability due to inherent variations within each animal. Non-invasive&#160;in vivo&#160;optical imaging provides a more fitting approach to monitor i.p. tumor burden in live mice.&#160;
Several non-invasive imaging methods are currently used in pre-clinical research for the monitoring of tumor growth and therapeutic responses. These include computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), and optical imaging such as fluorescence and bioluminescence 7-12. CT is a transmission imaging process combining X-ray and computer technology. It produces a cross-section image of detected beams of high-energy photon, which passes through the body with different speed. US is a type of reflection image, which sends high-frequency sounds to the body creating sound waves that are reflected with different speed depending on tissue density and recognized by the computer to produce a visual image. MRI and PET are emission imaging modalities that use magnetic energy and nuclear particles, respectively to produce the image. MRI creates a strong magnetic field that induces cells to produce their own radio frequencies, which are used to create an image while PET requires a sensitive camera to detect the radioactivity of the administrated labeled 2-fluorodeoxy-D-glucose 7,9,11. Finally, optical imaging is based on the detection of the emission light of bioluminescent or fluorescent reporters or probes 9,12.
In this report, we describe the use of fluorescence, which offers a few advantages over the other types of imaging modalities. With fluorescence imaging, cells can be genetically engineered to express fluorescent proteins constantly without requiring the addition of a substrate or ligation-based probes, which are requisite for bioluminescence and magnetic resonance imaging, respectively. Fluorescence reporters also typically express a brighter signal thus allowing the use of a less sensitive detection method 8,12. In addition, with fluorescence imaging, it is possible to detect tumors smaller than 1 cm, which is not achievable with CT 7-9. Finally, in contrast to bioluminescence, fluorescence signal does not require an aerobic environment and hence the signal is not limited in hypoxic environments, which are usually occurring in the cores of large tumors 13.
However, as any other technology, fluorescent-based imaging methods have its disadvantages. One of which is the inability of the machine-generated low energy photons to penetrate to a sufficient depth. Thus, to minimize the amount of diffused tissue photons the animals should be imaged at different angles. We describe a protocol to establish an i.p. ovarian cancer in nude mice and an approach for i.p. tumor monitoring that provides whole animal imaging through rotation. The rotator angles the mouse to specific and repeatable positions decreasing the tissue interference that often occurs between the light source and the detector. This optimizes the visualization of smaller tumors that may otherwise be missed.

<table><tbody><tr><td>RPMI 1640 media</td><td>GIBCO, by Life Technologies</td><td>23400-021</td><td></td></tr><tr><td>fetal bovine serum</td><td>Gemini Bioproducts</td><td>100-106</td><td></td></tr><tr><td>T75 cell culture flasks</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Trypsin</td><td>GIBCO, by Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Schein</td><td>NDC 11695-6776-1</td><td></td></tr><tr><td>Alcohol pads</td><td>Fischer Scientific</td><td>06-669-62</td><td></td></tr><tr><td>1 ml syringe</td><td>Becton Dickinson</td><td>309602</td><td></td></tr><tr><td>25 gauge needle</td><td>Becton Dickinson</td><td>305122</td><td></td></tr><tr><td>synthetic absorbable suture</td><td>Covidien</td><td>SL-636</td><td></td></tr><tr><td>tissue adhesive</td><td>Vetbond</td><td>1469SB</td><td></td></tr><tr><td>surgical scissors</td><td>VWR</td><td>82027-584</td><td></td></tr><tr><td>surgical forceps</td><td>VWR</td><td>82027-386</td><td></td></tr><tr><td>hemostat</td><td>VWR</td><td>82027-422</td><td></td></tr><tr><td>Paclitaxel</td><td>Hospira, Inc.</td><td>NDC 61703-345-50</td><td></td></tr><tr><td>Ibuprofen</td><td>Walgreens</td><td></td><td>Children&#039;s Ibuprofen 100 (100 mg/5 ml)</td></tr><tr><td>Puralube Vet ointment</td><td>Pharmaderm</td><td></td><td></td></tr><tr><td>In vivo MS FX PRO</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>MI software</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>athymic nude mice</td><td>Harlan</td><td></td><td></td></tr></tbody></table>

murine model for non-invasive imaging to detect and monitor ovarian cancer recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

Non-invasive live imaging allows the monitoring of i.p. tumor progression from the same mice through time. Intra-uterine injection of mCherry-labeled F2 ovarian cancer cells using the described protocol generates visible i.p. tumors at day 5 and carcinomatosis at day 32 (Fig. 5). Figure 6 shows the correlation of the obtained images with carcinomatosis observed after sacrificing the mice.
Imaging using MARS allows the detection of i.p. tumors that will otherwise be missed if image is acquired from only one single plane. Figure 7 (top panel) shows that even in Control mice with significant tumor burden, tumor size can be underestimated depending on the angle the image was taken from. The ability to ensure that tumor mass is not missed or underestimated is even more important at the conclusion of 1st round chemotherapy when animals are categorized as either a complete responder, partial responder, or non-responder (Fig. 7 middle panel). Similarly, during maintenance therapy it is of utmost importance to detect very small recurrent tumors to properly designate days to recurrence, which defines progression-free interval (Fig. 7 middle panel).
Quantitative assessment of tumor burden is optimized through animal rotation. Rotation of the animal to angles that will minimize the depth excitation and emission light travels through will allow for the most accurate capture of photons from the fluorescence, which is indicative of the quantity of tumor cells and thus tumor burden. Hence, quantitation is optimized for specific tumors in the animal depending upon its position in the rotation sequence.
In presenting images of rotation data sets, it is important to assign the images a min/max image contrast range that will effectively display all tumor masses, while allowing for visual delineation of individual tumor masses. For this study, ideal contrast range was found to be a minimum of 50 counts and a max of 1,200 counts. These values can be seen as a guideline for other studies, however, optimal contrast ranges will differ from study to study depending on tumor burden level, fluorescent peptide expression levels in cells, imaging system configuration, and capture settings.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig1highres.jpg" width="600" /> 
Figure 1: F2 ovarian cancer cells stably express mCherry fluorescence. (A) Phase image; (B) fluorescence image; (C) overlay of A and B. Note that 100% of cells express the fluorescence reporter.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig2highres.jpg" width="600" /> 
Figure 2: Intra-uterine injection of ovarian cancer cells. (A) anesthesia is continuously administered via a nose cone; (B-E) skin is incised to locate and clamp the uterine horn; (F) cancer cells are injected slowly into the lumen of the uterus.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig3highres.jpg" width="600" /> 
Figure 3: (A) 2D imaging with a temperature controlled and close circuit ventilated transparent animal tray within the imaging chamber; (B, C) tray is fitted with nose cones to deliver anesthesia and can hold up to five mice.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig4highres.jpg" width="600" /> 
Figure 4: Representative window panels taken from the analysis software to assist in data analysis. Please see text for explanation.
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig5highres.jpg" width="600" /> 
Figure 5: Detection of mCherry fluorescence co-localized with X-ray in longitudinal imaging sequence. Three mice with i.p. tumor are followed through time to assess i.p. tumor progression. Note that tumor progression may vary. Figure is a representative image of 3 mice with different rates of tumor progression and therefore varying tumor burden through time.
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig6highres.jpg" width="600" /> 
Figure 6: Correlation between i.p. tumor burden (top panel) and fluorescence/X-ray overlay image (bottom panel). About 32 days post injection of F2-mCherry ovarian cancer cells, 2D-imaging is performed and mice sacrificed to correlate actual tumor burden with the acquired image. Red arrows point to tumor burden.
<img alt="Figure 7" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig7highres.jpg" width="600" /> 
Figure 7: Rotation data sets allowing multi-angle imaging and detection of tumors in Control (top panel), Paclitaxel treated (middle panel), and recurrent mice (bottom panel). Figure shows image of a single mouse per panel as it is rotated using the MARS system. Note that even in Control mice with significant tumor burden, tumor size can be underestimated depending on the angle the image was taken from.

Watch this Scientific Journal Video about Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence at JoVE.com

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

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

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Medicine

12.2K Views.  Yale University School of Medicine. We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer. 

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

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Cancer Research

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

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

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

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Immunology and Infection

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Biology

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

Source: Koya, Y. et al. <a href="https://www.jove.com/t/54353">Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells.</a> J. Vis. Exp. (2016)
This video describes the protocol for creating a murine experimental model to study tumor development and peritoneal metastasis. This ovarian cancer model is generated by the orthotopic inoculation of human epithelial ovarian cancer cells into the ovaries of a female mouse.

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

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Chapters in this video

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

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

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells