Name: quantitation of intra-peritoneal ovarian cancer metastasis
Uploaded: 2016-07-18T14:00:00
Description: Watch this Scientific Journal Video about Quantitation of Intra-peritoneal Ovarian Cancer Metastasis at JoVE.com

This research was supported by research grants RO1CA109545 and RO1CA086984 to M.S.S. by the National Institutes of Health/National Cancer Institute and by an award from the Leo and Ann Albert Charitable Trust (to M.S.S.).

All in vivo studies were approved by the University of Notre Dame Animal Care and Use Committee and used female C57/BL6J mice.
1. Murine Ovarian Cancer Cell Culture
<ol>
	<li>Make the ID8 murine ovarian cancer cell culture medium as follows: 1 L of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 4% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin, 5 &#181;g/ml Insulin, 5 &#181;g/ml Transferrin and 5 ng/ml Sodium Selenite.</li>
	<li>Transduce ID8 murine ovarian canc...

<ol>
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In contrast to studies using human ovarian cancer cells that must be conducted in immunocompromised mice, the protocol described above utilizes immunocompetent C57/Bl6 mice and syngeneic murine ovarian cancer cells. While this enables assessment of the potential role of immune infiltrates in tumor progression and metastasis, the presence of dark hair on the abdominal surface renders imaging less sensitive. Use of a depilatory to remove hair prior to imaging enhances image acquisition, but is time-consuming, particularly ...

Epithelial ovarian cancer (EOC) is the most common cause of death from gynecologic malignancy, with an estimated 21,290 new diagnoses in the U.S. in 2015 and an estimated 14,180 deaths1. The vast majority (&#62; 75%) of women are diagnosed with late-stage disease (stage III or IV) characterized by diffuse intra-peritoneal metastasis and poor prognosis. Disease recurrence in the peritoneal cavity following first-line chemotherapy is also common and represents a major cause of mortality2,3. EOC metastasizes by an unique mechanism involving both direct extension from the primary tumor to neighboring peritoneal organs as well as by dissociation or shedding of cells from the primary tumor surface as single cells or multi-cellular aggregates. Cells are shed into the peritoneal cavity, wherein they resist detachment-induced apoptosis4. Buildup of peritoneal ascites is common, as shed tumor cells block peritoneal lymphatic drainage and tumors produce growth factors that alter vascular permeability. A portion of shed tumor cells attach to the surface of peritoneal organs and structures including intestine, liver, omentum and mesentery, whereupon they anchor and proliferate to produce multiple widely disseminated secondary lesions3,5. Hematogenous metastasis is uncommon. Thus, clinical management commonly consists of cytoreductive surgery including &#34;optimal debulking&#34;, defined as resection of all visible tumor (no matter how small). Complete cytoreduction is associated with a significant increase in overall survival6,7 and is associated with the challenge of identification and removal of lesions &#60; 0.5 cm.
Small animal models have proven utility in ovarian cancer research in improving our understanding of disease progression as well in identification of prognostic biomarkers and testing of novel chemotherapies or combination therapy approaches. As the primary site of ovarian cancer incidence and metastasis is the peritoneal cavity, orthotopic models of EOC metastasis involve the analysis and characterization of intraperitoneal disease. Although there have been recent improvements in the ability to image tumor cells, even at the single cell level, there still exist significant difficulties in quantifying the metastatic tumor burden of EOC. These challenges arise due to the number, size and anatomic location of metastatic lesions. Furthermore there exists a need to label cancer cells to distinguish them from normal host cells. Previous studies have utilized antibody-based labeling protocols or transfection of tumor cells with luciferase8,9. Direct fluorescent labeling of cancer cells was first reported by Chishima and coworkers in 199710. Fluorescent labels do not require addition of exogenous substrate and provide exquisite tumor cell specificity, providing a more effective means to track cancer metastasis11,12.
Herein we describe an optical imaging method for quantitative analysis of metastatic disease using a syngeneic orthotopic xenograft model comprised of red fluorescent protein (RFP)-tagged murine ID8 ovarian cancer cells13 and immuno-competent C57/Bl6 mice. We demonstrate a novel method of relative tumor burden quantification combining in vivo and ex vivo imaging with removal of tissue auto-fluorescence. This approach has potential utility in studies designed to evaluate the effect of specific genetic, epigenetic or micro-environmental modifications and/or treatment modalities on organ-specific metastasis of ovarian cancer.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:183px;">Corning</td>
			<td style="width:117px;">10-014-CM</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin/streptomycin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Insulin-transferrin-sodium selenite media supplement</td>
			<td>Sigma</td>
			<td>I-1884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Xtreme small animal imaging system</td>
			<td>Bruker Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Multispectral software</td>
			<td>Bruker Corp</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">lentiviral particles with Red fluorescent protein</td>
			<td>GenTarget, Inc.</td>
			<td>LVP023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin for cell culture</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">depilatory cream (such as Nair Hair Remover Lotion)</td>
			<td>purchases from drugstore&#160;</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software&#160;</td>
			<td>http://imagej.nih.gov/ij/&#160;</td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (forceps)</td>
			<td>Roboz Surgical Instrument</td>
			<td>&#160;RS 5130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (Scissors)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS 5910</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The metastatic mechanism of ovarian cancer is characterized by highly diffuse intra-peritoneal metastasis comprised of numerous lesions of varying size, including multiple small (&#60; 2mm) lesions. Thus, use of RFP-labelled tumor cells (Figure 1) and optical imaging provides an alternative method to manual counting and measurement of lesion size. The development of tumor burden over time can be determined by weekly weighing of mice and measurement of abdominal girth to g...

Watch this Scientific Journal Video about Quantitation of Intra-peritoneal Ovarian Cancer Metastasis at JoVE.com

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

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 ...

The overall goal of this experiment is to determine the relative tumor burden of intra-peritoneal ovarian cancer metastasis in a syngeneic orthotopic xenograft murine model. This novel method to image and quantify relative tumor burden can help us answer key questions in regulation of ovarian cancer metastasis. The fundamental advantage of this technique is that by un-mixing of fluorescent spectra, tissue auto fluorescence is removed from the images, enabling the measurement of standardized, relative tumor burden.This technique lends insights in the human ovarian cancer metastasis with implications towards monitoring the efficacy of therapeutics. Visual demonstration of this method is critical because we are coupling a robust imaging technique with an analytical procedure to extract quantitative data from the images. Demonstrating the procedure today will be Yueying Liu, the laboratory Program Manager for the Stack Lab.After 10 weeks of tumor cell growth, place the body of each anesthetized mouse into a small animal optical imaging system, one at a time. Scan all mice in the cohort at each time point. To observe fluorescently labeled tumor cells, execute a multi-spectral acquisition to collect a total of five images.Select a standard exposure of 15 seconds, two by two binning, a field of view of 16 centimeters, and an F stop of 1.1. Next, acquire a reflectance image of the entire animal using white light from an open excitation filter, an open emission filter, a standard exposure of 0.2 seconds with two by two binning, and a field of view of 16 centimeters, with an F stop of 2.8. Following euthanasia, use pointed dissection scissors to cut the skin anteriorly along the ventral mid-line of the mouse, from the top of the thighs to the bottom of the rib cage.Gently separate the skin from the peritoneal tissue, being sure not to puncture the peritoneal wall. Then cut laterally, both along the bottom of the rib cage, and the top of the thighs, to create two flaps of skin. Place the mouse on its ventral side in the scanner, so that the peritoneal cavity is facing downward with the skin flaps pulled open.Scan the mouse with its organs in situ, using the same parameters that were previously used for the live mouse. Now, continue dissecting the mouse by extracting the liver, the omentum, the pancreas, the stomach, the diaphragm, and the small and large intestines using standard techniques. Also, remove the left and right peritoneum, the ovaries, the kidneys, the mesentery, and lastly, the fat pad.Observe, enumerate, and record each of the visible tumors on the organs. Use a caliper to measure the diameter of each tumor. Designate tumors that are less than two millimeters in diameter as small, in between two and five millimeters as medium, and greater than five millimeters as large.Next, place the organs on the organ scanning template, found in Figure Four of the accompanying text protocol, and use it to organize the location of each organ during the next scan. Use PBS to keep the organs moist. Scan each sheet of organs using the small animal optical imaging system and the parameters that were previously used.After scanning, place the organs in 10%formaldehyde, and process them for histology using standard techniques. In order to reduce the amount of auto fluorescence in each multi-spectral scan, first load the red fluorescent protein in vivo spectral file followed by the auto floor in vivo red file in the unmixed images window, and select un-mix. Then export the dot bip files in a 16 bit unscaled dot tif format, and load them into Image J, by going to file, open, and then selecting the correct 16 bit dot tif file.Next, go to the image menu, and select adjust, then brightness contrast, and set the minimum displayed value to zero, and the maximum displayed value to plus 35353. Then under image menu again, go to look up tables, and select red hot. Various animal models will require different brightness levels, but it is important to keep the brightness consistent throughout a given study.Using the free hand selection tool, draw a free form region of interest selection around each organ. The free form region of interest should be drawn close to the organ borders. Then, type control and M to use the measure tool to calculate the surface area of each organ.Record this data in a spread sheet. With the region of interest that was drawn around each organ, right click and select duplicate to include only the organ. Then, under image, go to adjust, then threshold, and set the lower threshold level to plus 1, 200 and the upper threshold level to plus 1, 700.Also, check the dark background, and then select okay. This will select only the most brightly fluorescing regions. The images may require manual manipulation of the threshold sliders in Image J so that the previously brightest areas are selected for the analysis step, as shown above.Different disease models will require different threshold constraints, but it is important to keep the threshold levels consistent throughout a given study. Under analyze, select analyze particles, and change the size of the pixel squared to 10 to infinity. Choose to show outlines, and check to select only display results, and then click okay.This will measure both the areas, and raw integrated densities of the bright regions deemed tumors. Record the values of the surface area and the raw integrated density of each tumor selection together in the same spread sheet containing the organ surface areas. Then, under analyze, select tools, and go to calibration bar.Add a calibration scale bar to the images of the organs. Montages can contain greater or fewer numbers of organs as dictated by a given study. Next, under file, go to save as, and save the montage as both a dot tif and a dot jpeg.Then, open the dot jpeg file of the organs, and go to the insert menu to add a text box to each image. Add organ labels by creating a text box below each of the three rows of organs. Open each of the 16 bit dot tif files of the full body scans into Image J in order of mouse number.Then, go to the image menu, and select adjust, and then brightness contrast. Set the minimum displayed value to zero, and the maximum displayed value to plus 35353. Next, go back to the image menu, and to look up tables, and then select red hot.Once this is complete, save the montage as both a dot tif and a dot jpeg file. The ID8 murine ovarian cancer cell line that is injected into the mice 10 weeks prior to imaging fluoresces in the red channel, and continues to do so throughout growth. Live imaging using a small animal optical imaging system is able to view the expanding ID8 cells in the mouse and provides a more accurate approach than simply weighing the mouse in order to asses tumor burden.Following euthanasia and removal of the ventral skin layer, the intact organs can be viewed in more detail. However, individual organs placed on the organ scanning template display the tumor burden clearly in each organ. The tumor burden in the omentum and pancreas, ovaries and mesentery of the mouse shown on the left is much greater than those same organs in the mouse shown on the right.The observation of differential tumor burden is confirmed quantitatively both in terms of tumor service area and tumor signal intensity. Once mastered, this technique can be used to image up to 18 living mice per hour. Dissection and ex vivo imaging of individual organs takes roughly 20 minutes per specimen.Following this procedure, other methods like quantitative magnetic resonance can be performed in order to address hypotheses such as the impact of obesity on ovarian cancer metastasis, as shown in our 2015 cancer research article.

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