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 ...

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