The authors thank the members of the Department of Obstetrics and Gynecology and Cancer Biology for their helpful discussions and technical assistance. This study was supported by a Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (15K15604; to H. Kajiyama).

The treatment protocol follows the guidelines for animal experimentation adopted by Nagoya University.
1. Preparation of Cell Suspensions
<ol>
	<li>Human ovarian clear cell carcinoma cell line.
	<ol>
		<li>Maintain ES-2 cells12 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 humidified incubator.</li>
		<li>Collect cells in the logarithmic growt...

<ol>
	<li>Jemal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics.">Global cancer statistics.</a> CA Cancer J Clin. 61 (2), 69-90 (2011).</li><li>Kajiyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+clinical+outcome+of+patients+with+recurrent+epithelial+ovarian+carcinoma:+is+it+the+same+for+each+histological+type.">Long-term clinical outcome of patients with recurrent epithelial ovarian carcinoma: is it the same for each histological type.</a> Int J Gynecol Cancer. 22 (3), 394-399 (2012).</li><li>Kikkawa, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+treatment+of+epithelial+ovarian+cancer.">Advances in treatment of epithelial ovarian cancer.</a> Nagoya J Med Sci. 68 (1-2), 19-26 (2006).</li><li>Yoshikawa, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinicopathologic+features+of+epithelial+ovarian+carcinoma+in+younger+vs.+older+patients:+analysis+in+Japanese+women.">Clinicopathologic features of epithelial ovarian carcinoma in younger vs. older patients: analysis in Japanese women.</a> J Gynecol Oncol. 25 (2), 118-123 (2014).</li><li>Kajiyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+SDF-1alpha/CXCR4+axis+in+the+enhanced+peritoneal+metastasis+of+epithelial+ovarian+carcinoma.">Involvement of SDF-1alpha/CXCR4 axis in the enhanced peritoneal metastasis of epithelial ovarian carcinoma.</a> Int J Cancer. 122 (1), 91-99 (2008).</li><li>Terauchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+involvement+of+TWIST+in+enhanced+peritoneal+metastasis+of+epithelial+ovarian+carcinoma.">Possible involvement of TWIST in enhanced peritoneal metastasis of epithelial ovarian carcinoma.</a> Clin Exp Metastasis. 24 (5), 329-339 (2007).</li><li>Bronstein, L., Zechner, C., Koeppl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+inference+of+reaction+kinetics+from+single-cell+recordings+across+a+heterogeneous+cell+population.">Bayesian inference of reaction kinetics from single-cell recordings across a heterogeneous cell population.</a> Methods. 85, 22-35 (2015).</li><li>Guo, W., Zhang, S., Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+orthotopic+model+of+breast+cancer+metastasis+to+the+lung.">Establishment of a novel orthotopic model of breast cancer metastasis to the lung.</a> Oncol Rep. 33 (6), 2992-2998 (2015).</li><li>Lewis-Tuffin, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Src+family+kinases+differentially+influence+glioma+growth+and+motility.">Src family kinases differentially influence glioma growth and motility.</a> Mol Oncol. , (2015).</li><li>Saar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+tumorgrafts+in+nude+mice:+A+new+method+to+study+human+prostate+cancer.">Orthotopic tumorgrafts in nude mice: A new method to study human prostate cancer.</a> Prostate. 75 (14), 1526-1537 (2015).</li><li>Zou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-29c+suppresses+pancreatic+cancer+liver+metastasis+in+an+orthotopic+implantation+model+in+nude+mice+and+affects+survival+in+pancreatic+cancer+patients.">miR-29c suppresses pancreatic cancer liver metastasis in an orthotopic implantation model in nude mice and affects survival in pancreatic cancer patients.</a> Carcinogenesis. 36 (6), 676-684 (2015).</li><li>Lau, D. H., Lewis, A. D., Ehsan, M. N., Sikic, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifactorial+mechanisms+associated+with+broad+cross-resistance+of+ovarian+carcinoma+cells+selected+by+cyanomorpholino+doxorubicin.">Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin.</a> Cancer Res. 51 (19), 5181-5187 (1991).</li><li>Bao, L., Matsumura, Y., Baban, D., Sun, Y., Tarin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+inoculation+site+and+Matrigel+on+growth+and+metastasis+of+human+breast+cancer+cells.">Effects of inoculation site and Matrigel on growth and metastasis of human breast cancer cells.</a> Br J Cancer. 70 (2), 228-232 (1994).</li><li>Huang, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiological+investigation+on+major+depressive+disorder+in+the+most+heavily+damaged+areas+from+Wenchuan+earthquake+in+2008.">Epidemiological investigation on major depressive disorder in the most heavily damaged areas from Wenchuan earthquake in 2008.</a> Zhonghua Liu Xing Bing Xue Za Zhi. 31 (2), 167-170 (2010).</li><li>Ma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+of+neonatal+respiratory+failure+related+to+socioeconomic+factors+in+Hebei+province+of+China.">Mortality of neonatal respiratory failure related to socioeconomic factors in Hebei province of China.</a> Neonatology. 100 (1), 14-22 (2011).</li><li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+levels+of+FGF-21+are+increased+in+coronary+heart+disease+patients+and+are+independently+associated+with+adverse+lipid+profile.">Serum levels of FGF-21 are increased in coronary heart disease patients and are independently associated with adverse lipid profile.</a> PLoS One. 5 (12), e15534 (2010).</li><li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumbar+interspinous+non-fusion+techniques:+comparison+between+Coflex+and+Wallis.">Lumbar interspinous non-fusion techniques: comparison between Coflex and Wallis.</a> Nan Fang Yi Ke Da Xue Xue Bao. 30 (11), 2455-2458 (2010).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wave+intensity+analysis+of+carotid+artery:+a+noninvasive+technique+for+assessing+hemodynamic+changes+of+hyperthyroid+patients.">Wave intensity analysis of carotid artery: a noninvasive technique for assessing hemodynamic changes of hyperthyroid patients.</a> J Huazhong Univ Sci Technolog Med Sci. 30 (5), 672-677 (2010).</li><li>Song, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptional+profiling+and+preliminary+study+on+heterosis+mechanism+of+super-hybrid+rice.">Comparative transcriptional profiling and preliminary study on heterosis mechanism of super-hybrid rice.</a> Mol Plant. 3 (6), 1012-1025 (2010).</li><li>Wan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+variation+of+Han+female+adolescent+bone+development+in+Henan+and+Zhejiang+province.">The analysis of variation of Han female adolescent bone development in Henan and Zhejiang province.</a> Fa Yi Xue Za Zhi. 26 (2), 97-99 (2010).</li><li>Wang, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitation-emission+fluorescence+characterization+study+of+the+three+phenolic+compounds.">Excitation-emission fluorescence characterization study of the three phenolic compounds.</a> Guang Pu Xue Yu Guang Pu Fen Xi. 30 (5), 1271-1274 (2010).</li></ol>

Animal models are essential to analyze the mechanisms of tumor development, progression, metastasis, and drug efficacy against cancer cells. Mice bearing human ovarian cancer cells also have been used as an animal model. Here, we describe a less invasive procedure for the administration of ovarian tumor cells at an orthotopic site. Using this procedure, inoculation into the ovary through a retroperitoneal approach from the dorsal flank offers significant advantages over other commonly used techniques. Firstly, orthotopic...

Epithelial ovarian carcinoma (EOC) accounts for the highest rate of cancer-related mortality among gynecological malignancies1. EOC is associated with a poor prognosis, primarily due to its late symptomatology, and is often associated with multiple intraperitoneal disseminations and distant metastases2-4. Peritoneal dissemination is a multi-step process. Firstly, tumor cells detach from the primary lesions, and migrate into the abdominal cavity. When the tumor cells attach to the peritoneal mesothelium, they start to invade tissues through the mesothelium5,6. In order to better understand the tumor biology (e.g., cancer progression and therapeutic response), mouse models provide a wealth of information. A xenograft with human cancer cells is widely used for mouse models in which human cancer cells are inoculated subcutaneously, intraperitoneally, intravenously, and orthotopically. An animal model with an orthotopically grafted tumor can more efficiently and accurately generate results that reflect the tumor environment in humans in comparison with an animal model with a heterotopically grafted tumor. Therefore, for many human tumors, orthotopic transplantation models have been established7-11.
Here, we describe the orthotopic inoculation of human EOC cells through a retroperitoneal approach from the dorsal flank, which has limited invasiveness compared to ventral inoculation. This technique can provide a variety of useful information on EOC, especially the mechanism of intraperitoneal dissemination.

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Matrigel matrix&#160;</td>
			<td style="width:151px;">BD</td>
			<td style="width:163px;">354234</td>
			<td style="width:163px;">ECM-based hydrogel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin syringe</td>
			<td>TERUMO</td>
			<td>SS-05M2913</td>
			<td>1/2cc, 29Gx1/2&#34;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Suturing needle with suture</td>
			<td></td>
			<td></td>
			<td>11mm, 3/8, Nylon6-0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reflex 7mm Wound Clip Applier</td>
			<td>CellPoint Scientific</td>
			<td>204-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Reflex 7mm wound clips</td>
			<td>CellPoint Scientific</td>
			<td>203-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Six nude mice had their ovaries inoculated with the human clear cell carcinoma cell line ES-2, as described in this protocol. As shown in Figure 1A, after 2 weeks, 5 of the 6 mice had a tumor in the ovary inoculated with ES-2 cells, but there was no tumor in the contralateral ovary without inoculation (used as a control). Moreover, 2 of the 5 mice showed multiple peritoneal disseminations with ascites. Cells metastasize to the liver (Figure 1B). The ovari...

Watch this Scientific Journal Video about Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells at JoVE.com

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.

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 ...

The overall goal of this procedure is to observe the development of original and metastatic tumor formation in epithelial ovarian carcinoma.This method can help answer key questions in the gynecologic oncological field, such as understanding the tumor biology or peritoneal defemenation.The main advantage of this technique is that orospatic inoculation of human epithelial ovarian carcinoma, or EOC cells, is performed through a retroperitoneal approach grown with a dorsal flank.To prepare a human ovarian clear cell carcinoma cell line, plate and maintain ES2 cells in RPMI-1640 medium with 10%FBS and PenStrep at 37 degrees Celsius, and 5%CO2.In the logarithmic growth phase, remove the old medium from the culture and use three to four millimeters of PBS to wash the cells once.Next, add one millimeter of trypsin/EDTA and rock the vessel to cover the entire monolayer.Remove and discard the trypsin solution and return the cells to the incubator for three to five minutes.After examining the cells under a microscope to ensure the cells have detached and are floating, add five millimeters of fresh serum-containing growth medium to inactivate the trypsin and re-suspend the cells before transferring them to a 15-millimeter conical tube.Centrifuge the suspension at 300 times G and room temperature

murine-experimental-model-original-tumor-development-peritoneal

Research

JoVE Journal

Cancer Research

10.2K 조회수.  Bell Research Center for Reproductive Health and Cancer. The overall goal of this procedure is to observe the development of original and metastatic tumor formation in epithelial ovarian carcinoma.This method can help answer key questions in the gynecologic oncological field, such as understanding the tumor biology or peritoneal defemenation.The main advantage of this technique is that orospatic inoculation of human epithelial ovarian carcinoma, or EOC cells, is performed through a retroperitoneal approach grown with a dorsal flank.To prepare a hum...