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.
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/µl 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.
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.
The treatment protocol follows the guidelines for animal experimentation adopted by Nagoya University.
1. Preparation of Cell Suspensions
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.
3. Post-surgical Treatment
4. Analysis of Tumors and Metastases
NOTE: The tumor will be formed at the injected ovary 2 – 3 weeks after inoculation.
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 ovaries, both with the tumor mass at the inoculation site and on the control side, were dissected, sectioned, stained with hematoxylin-eosin (HE), and analyzed under a microscope (Figure 2). A large number of ovarian cancer cells were observed in the inoculated ovary, but not on the contralateral side. The cell morphology was the same as that of the parental tumor.
Figure 1: Tumor Formation by Orthotopic Inoculation with Human Clear Cell Carcinoma Cells ES-2. A and B, the BALB/c nu/nu mouse underwent orthotopic inoculation with ES-2 cells. After 2 weeks, the mouse was sacrificed for necropsy. B, Cells metastasize to the liver (as indicated by arrows). Please click here to view a larger version of this figure.
Figure 2: Hematoxylin-eosin Staining of Ovary with Orthotopic Inoculation with Human Clear Cell Carcinoma Cells ES-2. A and B, ovary with inoculation of ES-2 cells, stained with hematoxylin-eosin (HE). Similar staining was seen compared to that of clear cell carcinoma in the human ovary. C and D, HE staining of the ovary without inoculation as a control. Scale bars, 100 μm (A, C), 20 μm (B, D). Please click here to view a larger version of this figure.
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 inoculation can provide more efficient and accurate results that reflect the tumor environment in humans13,21. Secondly, this retroperitoneal approach from the dorsal flank only needs a small dissection (< 1 – 2 cm) so it is a rapid procedure compared to the peritoneal approach. Therefore, this retroperitoneal approach is less invasive for mice. Thirdly, the murine ovary is very small, and so unable to accept a large amount of reagents. In this presented procedure, we use highly concentrated cell suspensions (1 x 105 cells/µl) and a low inoculation volume (1 – 2 µl/injection) as a single shot; therefore, it minimizes damage to the ovary. It also avoids the leakage of inoculated cells from the ovary. Although further research is essential, the described murine orthotopic inoculation may be a useful approach to help develop new therapies for the treatment of EOC in the future.
There are two critical points in this protocol. Firstly, the cells are used in the logarithmic growth phase. Secondly, the cells suspended with ECM-based hydrogel are placed on ice until use. When the ECM-based hydrogel starts to warm, the gelation process will begin. To avoid the gelation of the ECM-based hydrogel, cells re-suspended with the ECM-based hydrogel and instruments (e.g., tubes, syringes, and needles) are kept on ice until use. There is one limitation of this technique. The injection volume of the cell suspension is less than 3 µl, because the mouse ovary cannot accept a large volume of inoculum.
This protocol can be modified in some points. If an in vivo imaging system is available, cells bearing enzymes that produce bioluminescence (e.g., luciferase) or fluorescent protein (e.g., GFP) can be used in this protocol. This modified protocol will provide useful information for the dynamics of the spread of cancer cells from the primary tumor. Also, this protocol uses ES-2 cells for inoculation with mouse ovary. ES-2 cells are derived from clear cell carcinoma, and this cell line exhibits aggressive growth in nude mouse. Other cell lines (e.g., serous carcinoma) also can be used this protocol instead of ES-2 cells.
The authors have nothing to disclose.
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).
Matrigel matrix | BD | 354234 | ECM-based hydrogel |
Insulin syringe | TERUMO | SS-05M2913 | 1/2cc, 29Gx1/2" |
Suturing needle with suture | 11mm, 3/8, Nylon6-0 | ||
Reflex 7mm Wound Clip Applier | CellPoint Scientific | 204-1000 | |
Reflex 7mm wound clips | CellPoint Scientific | 203-1000 |