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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Cell Suspensions
<ol>
	<li>Human ovarian clear cell carcinoma cell line
	<ol>
		<li>Maintain ES-2 cells&#160;in RPMI-1640 medium with 10% fetal bovine serum (FBS) and penicillin/streptomycin (penicillin: 100 units/mL, streptomycin: 0.1 mg/mL). Culture cells at 37 &#176;C in a 5% CO2&#160;humidified incubator.</li>
		<li>Collect cells in the logarithmic growth phase in 0.05% trypsin/0.02% EDTA solution (trypsin/EDTA).
		<ol>
			<li>First, remove the old media from the cell culture vessel, then wash the cells once with PBS (PBS without Ca2+/Mg2+,3-4 mL per 25 cm2).</li>
			<li>Next, add 1 mL per 25 cm2 of trypsin/EDTA. Rotate the cell culture vessel to cover the cell monolayer with trypsin/EDTA. After removing and discarding trypsin/EDTA, return the cell culture vessel to the incubator for 3-5 min or until cells are detached.</li>
			<li>Examine the cells under a microscope to ensure the cells are detached from the surface of the cell culture vessel and floating.</li>
			<li>Add fresh serum-containing growth medium to inactivate the trypsin (5 mL per 25 cm2) and resuspend the cells. Transfer resuspended cells to a 15 mL conical tube.</li>
			<li>Centrifuge at 300 x g for 5 min. Perform centrifugation either at 4 &#176;C or room temperature. Carefully remove supernatant and resuspend cells with growth medium (5 mL per 25 cm2).</li>
		</ol>
		</li>
		<li>Count cells (e.g., by trypan-blue exclusion), then re-suspend in growth medium at a density of 2 x 108 cells/mL. Put the suspended cells on ice.</li>
	</ol>
	</li>
	<li>Thaw frozen extracellular matrix (ECM)-based hydrogel aliquots in an ice bath.</li>
	<li>Add the cell suspension to ECM-based hydrogel aliquots at a ratio of 1:1 (final cell concentration: 1 x 105&#160;cells/&#181;L of medium-ECM-based hydrogel) and mix thoroughly. Place the prepared cell suspensions in an ice bath until use.</li>
</ol>
2. Orthotopic Inoculation with Cell Suspensions
NOTE: Surgery does not require a dedicated suite. Surgery can be performed on a laboratory benchtop in an area of the room that is separate and has minimal activity. A laminar hood also may be used. Sterile surgical instruments must be used, and scissors should not be used to make skin incisions.
<ol>
	<li>Use female nude mice (BALB/c nu/nu) at 8 weeks of age for this orthotopic inoculation model.</li>
	<li>First, anesthetize the mouse using inhalation with isoflurane (2-4%). Check anesthetic depth by verifying lack of withdrawal upon firm toe pinch. After the mouse is anesthetized, apply a bland sterile ophthalmic ointment to the eyes to prevent drying as necessary.</li>
	<li>Place the anesthetized mouse on an operating stage, belly-side down. Disinfect the surgical area several times with both alcohol and an iodine-based or chlorhexidine-based scrub.</li>
	<li>Make a 2-cm incision vertically near the spine between the right costal arch and femur. Use a sterile surgical drape around the incision site. Fix the incised skin surfaces with clamps to maintain the opening. 
	NOTE: Retractors can be used instead of clamps.</li>
	<li>Next, make a second incision (approximately 1 cm) at the parietal peritoneum under the first incision to open the abdominal cavity. Clamp the incised abdominal surfaces. Clip the fat tissue that surrounds the ipsilateral ovary and fallopian tube with a clamp to remove the ovary from the abdominal cavity. Carefully place the ovary and fallopian tube on gauze. Then, place the mouse belly-side down under the dissecting microscope.</li>
	<li>Place the cell suspension in an insulin syringe (1/2 cc, 29 G) just before injection. Carefully inject the prepared cell suspension (1-2 &#181;L) into the ovary. For several seconds, retain the needle within the ovary to ensure gelation of the ECM-based hydrogel. After gelation, the injected tumor cells remain inside the ovary.</li>
	<li>Carefully return the ovary into the body. Suture the second incision with sterile medical silk and then seal the skin with wound clips.</li>
	<li>After surgery, the mice will be administered analgesia (e.g., meloxicam, intramuscular or subcutaneous, 0.2 mg/kg) for initial 24 hr&#160;period.</li>
</ol>

<table><tbody><tr><td>Matrigel matrix</td><td>BD</td><td>354234</td><td>ECM-based hydrogel</td></tr><tr><td>Insulin syringe</td><td>TERUMO</td><td>SS-05M2913</td><td>1/2 cc, 29 G x 1/2&quot;</td></tr><tr><td>Suturing needle with suture</td><td></td><td></td><td>&amp;nbsp;11 mm, 3/8, Nylon6-0</td></tr><tr><td>Reflex 7 mm Wound Clip Applier</td><td>CellPoint Scientific</td><td>204-1000</td><td></td></tr><tr><td>Reflex 7 mm wound clips</td><td>CellPoint Scientific</td><td>203-1000</td><td></td></tr></tbody></table>

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.

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

Orthotopic inoculation helps deliver cancer cells to a recipient animal at a specific tissue or site matching the cells&#39;&#160;natural anatomical location. To begin, prep an anesthetized female nude mouse, belly side down, on an operating stage. Make a vertical incision near its spine at a position between the rib cage and the femur. Open the abdominal muscles to expose the peritoneal cavity partially. Locate one of the ovaries.
Prepare a suspension of ovarian cancer cells with chilled extracellular matrix in a syringe. The low temperature prevents the suspension from solidifying. Inject this suspension into the mouse&#39;s ovary. Hold the needle in place to allow the matrix to solidify and embed the cancer cells within the ovary. Retract the needle. Suture the incision to close the wound. Allow the mouse to recover.
Over time, the injected ovarian cancer cells proliferate to form a primary tumor. Eventually, a few cells detach from their primary location and disseminate within the peritoneal cavity. When these migrating tumor cells attach to the peritoneal cavity lining, they invade the peritoneal tissue and proliferate to form metastasized secondary tumors.

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2.4K Views. Source: Koya, Y. et al. Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells. 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. 

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

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

JoVE Journal

Medicine

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 ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

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

Transcript

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

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity