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 cancer cells13 to express Red Fluorescent Protein (RFP) using commercially procured lentiviral particles expressing RFP and a selection marker (blasticidin gene). Note that green fluorescent protein can be used, but provides a weaker fluorescence signal.
	<ol>
		<li>Immediately prior to transduction, culture cells to 50 - 70% confluence and add fresh medium (without penicillin/streptomycin) that includes 1 ml polybrene (5 &#181;g/ml final concentration).</li>
		<li>Gently pipette RFP-lentivirus (20 &#181;l) dropwise into the dish and incubate at 37 oC for 72 hr. Remove medium and incubate with fresh medium for 24 hr. Begin selection of transduced cells by adding blasticidin to the culture medium (5 &#181;g/ml) and monitor for red cells using fluorescence microscopy (Figure1).</li>
	</ol>
	</li>
	<li>Culture the RFP-tagged cells in ID8 medium by seeding approximately 106 cells into a tissue culture dish and visualizing daily using a light microscope until the cell monolayer is confluent.</li>
	<li>When confluent, harvest the cells using trypsin (0.25%, 3 min) and return the flask to the 37 oC incubator until the cells are detached.</li>
	<li>Neutralize the trypsin with 6 ml of ID8 culture medium containing serum. Pipette up and down against the bottom of the dish, then transfer to a 15 ml conical tube. Centrifuge at 1,200 RPM for 2 min, then aspirate the medium using a glass pipette.</li>
	<li>Wash the cells with Phosphate Buffered Saline (PBS) by gently resuspending in 6 ml warm PBS and centrifuging as in 1.5 above. Re-suspend in PBS to a final concentration of 5 x 106 cells/ml. Keep cells warm (37 oC) until injection.</li>
</ol>
2. Intra-peritoneal Injection of ID8 Cells and in vivo Imaging
<ol>
	<li>Inject each mouse with 2 mL of the warm ID8-PBS solution for a total of 10 million cells through intra-peritoneal (i.p.) injection. Allow tumor cells to grow in vivo for approximately 10 weeks with weekly live imaging.</li>
	<li>Immediately following injection and each week thereafter, weigh the mice and then anesthetize each mouse using isofluorane (2.5% flow rate in 0.5 L/min O2 flow). Note:&#160;The mice were weighed, not as a quantitative measure of tumor burden, but rather a more general measure of individual mouse physical progression throughout the study.&#160; Mice weights were compared with their own previous weight values. &#160;
	<ol>
		<li>Anesthetize mice by placing in an isofluorane chamber. Maintain constant anesthesia while scanning by positioning the mouse head in the nose-cone for exposure to 2.5% flow rate isofluorane throughout the procedure.</li>
	</ol>
	</li>
	<li>Use depilatory cream to remove the hair on the torso and abdomen of each mouse, as the black hair will cause attenuation of the fluorescence signal. Mice are bathed with warm water after application of the depilatory cream and dried with RT paper towels. &#160;</li>
	<li>While anesthetized, scan the entire body of each mouse using a small animal optical imaging system. Scan all mice in a cohort at each time point. Use the following two step protocol (Figure 2A):
	<ol>
		<li>In Step 1, to observe fluorescently labeled (RFP) tumor cells, execute multispectral acquisition to collect a total of 5 images using excitation filters of 440 nm, 460 nm, 480 nm, 520 nm and 540 nm, and an emission filter of 600 nm. Use the following acquisition parameters: a standard exposure of 15 sec, 2 x 2 binning, FOV of 160 mm, and fstop of 1.1.</li>
		<li>In Step 2, to observe the whole animal, acquire a reflectance image using an open excitation filter (white light), an open emission filter, a standard exposure of 0.2 sec with 2 x 2 binning and an FOV of 16 cm. &#160;Note: The actual imaging time is ~1 min.&#160;</li>
	</ol>
	</li>
</ol>
3. Mouse Dissection and Image Acquisition
<ol>
	<li>When live imaging shows the presence of extensive intra-peritoneal disease or animals exhibit accumulation of ascites (as evidenced by abdominal distension), loss or gain of body weight greater than 20%,&#160;lethargy or other signs of distress, euthanize each mouse using CO2 anesthesia followed by cervical dislocation.</li>
	<li>Cut the skin anteriorly along the ventral midline of the mouse, using pointed dissection scissors, from the top of the thighs to the bottom of the rib cage. Take extreme care to cut through only the skin layer (Figure 2Bi). Gently separate the skin from the peritoneal tissue, being sure to not puncture the peritoneal wall.</li>
	<li>Using pointed dissection scissors, cut laterally both along the bottom of the rib cage and the top of the thighs to create two flaps of skin (Figure 2B ii,iii).</li>
	<li>Place each mouse on its ventral side (peritoneal cavity facing downward) with the skin flaps pulled open and scan each mouse with its organs in situ using the small animal optical imaging system (Figure 3A,B).
	<ol>
		<li>Use the same image acquisition parameters as described in 2.4.1.-2.4.2.</li>
	</ol>
	</li>
	<li>Continue dissecting the mouse by extracting individual peritoneal organs including liver, omentum/pancreas, stomach, diaphragm, small intestine, large intestine, left and right peritoneum, ovaries, kidneys, mesentery and fat pad.
	<ol>
		<li>Observe, enumerate, and record visible tumors on each organ. Use a caliper to measure the diameter of each tumor.</li>
		<li>Designate tumors less than 2 mm in diameter as small, in between 2 and 5 mm as medium and greater than 5 mm as large.</li>
	</ol>
	</li>
	<li>Place the organs on the organ scanning template&#160;(Figure 4) that identifies a predetermined location for each organ. Use PBS to keep the organs moist.</li>
	<li>Scan each sheet of organs using the small animal optical imaging system (Figure 3C,D).
	<ol>
		<li>Use the same image acquisition parameters as described in step 2.4.</li>
	</ol>
	</li>
	<li>After scanning, place organs in 10% formaldehyde to enable subsequent processing for histology.</li>
</ol>
4. Quantification of Tumor Burden in the Fluorescence Images
<ol>
	<li>In order to reduce the amount of auto-fluorescence in each multispectral scan, first unmix the files using software available with the small animal imaging system.
	<ol>
		<li>Load first the RFP: In Vivo spectral file, followed by the Autofluor: In Vivo Red file in the Unmixed Images window and select Unmix.</li>
	</ol>
	</li>
	<li>Export the .bip files as 16 bit .tiff (unscaled) format .</li>
	<li>Use ImageJ software (ex vivo organs and then the full mouse body with organs in situ).
	<ol>
		<li>Use File &#62; Open to open the 16 bit .tiff files of the organ scanning template files in ImageJ.</li>
		<li>Under Image &#62; Adjust &#62; Brightness/Contrast, choose to Set the Minimum displayed value to 0 and the Maximum displayed value to +35353 and then under Image &#62; Lookup Tables, select Red Hot.&#160;Note: Various animal models will require different brightness levels, but it is important to keep the brightness consistent throughout a given study.</li>
		<li>Using the Freehand Selection tool, draw a free-form region of interest selection (ROI) around each organ and use the Measure tool (CTRL+M) to calculate the surface area of each organ; record the surface Area of each organ in a spreadsheet.</li>
		<li>With the ROI drawn around each organ, right click within the ROI and select Duplicate to include only the organ.</li>
		<li>While looking at the individual organ, under Image &#62; Adjust &#62; Threshold, choose to Set the threshold of the image to a Lower Threshold Level of + 1200 and an Upper Threshold Level of + 1700 to select only the most brightly fluorescing regions and check to select Dark Background; select OK.&#160;Note: The images may require manual manipulation of the threshold sliders in ImageJ so that the previously brightest areas are selected for the analysis step, as shown above.&#160; Different disease models will require different threshold constraints, but it is important to keep the threshold levels consistent throughout a given study.</li>
		<li>Under Analyze &#62; Analyze Particles, change the Size (pixels ^ 2) to 10-Infinity, choose to Show: Outlines, check to select only &#34;Display results&#34; and click OK to measure both the areas and raw integrated densities of these bright regions, deemed tumors.</li>
		<li>Record the values of the surface Area and the Raw Integrated Density of each tumor selection displayed in the Results table in the same spreadsheet containing the organ surface areas.</li>
		<li>Under Analyze &#62; Tools &#62; Calibration Bar, add a calibration scale bar to the images of the organs. Note: In this example, this was done by changing the Location to Upper Right, the Fill Color to Black, the Label Color to White, the Number of Labels to 5, the Decimal Places to 0, the Font Size to 12 and the Zoom Factor to 4.0 and enabling Bold Text.</li>
		<li>Under File &#62; Save As&#8230;, save the montage as a .Tiff and .Jpeg.</li>
		<li>Use File &#62; Open to open the .jpeg file of the organs and then under Insert &#62; Text Box, add organ labels by creating a text box below each of the three rows of organs.</li>
		<li>Use File &#62; Open to open each of the 16 bit .tiff files of the full body scans in ImageJ in order of mouse number.</li>
		<li>Under Image &#62; Adjust &#62; Brightness/Contrast, choose to Set the Minimum displayed value to 0 and the Maximum displayed value to + 35353 and then under Image &#62; Lookup Tables, select Red Hot.</li>
		<li>Under File &#62; Save As&#8230;, save the montage as a .Tiff and .Jpeg.</li>
	</ol>
	</li>
</ol>
5. Data Analysis
<ol>
	<li>Add the tumor selection areas and raw integrated densities, respectively, for each organ of each mouse and record the total organ tumor area for each mouse.</li>
	<li>Normalize the recorded area and raw integrated density values for the size of each organ by dividing the total tumor area and total raw integrated density values by the corresponding organ surface area (Table 1).</li>
	<li>Calculate and plot the average of both the normalized tumor surface areas and the normalized raw integrated density values (Figure 5A,B).</li>
	<li>Compare these average values to those obtained by visually counting the tumors that were visible to the naked eye during the inspection of each organ for metastatic lesions.</li>
</ol>

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This article is part of a special issue on Multimodal Pre-Clinical Imaging, sponsored by Bruker Biospin.

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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10.8K Views.  University of Notre Dame. 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.

Video: Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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.

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 ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...