Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.
Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.
I. Preparation of Ovarian Tumor Cells
II. Intrabursal Injection
This procedure requires assistance from a second person. All surgical procedures are conducted under aseptic conditions. This includes wearing surgical attire and using sterile surgical instruments, syringe, and needles.
III. Oral Gavage Administration
IV. In Vivo Imaging
We use the Caliper Life Sciences to monitor the behavior of cells injected into intrabursal cavity. Experiments using this system typically have a timeframe of 4~16 weeks from the time of tumor implantation.
V. Representative Results
Figure 1. In Vivo imaging of ovarian tumor cells in an orthotopic mouse model. OVCAR5 cells expressing luciferase were injected intrabursally into right ovary and imaged over time. Images were taken 13 (left), 17 (middle), and 22 (right) days after the injection using the IVIS Spectrum imaging system. Dorsal side is shown in upper panel and ventral side is in lower panel. Note that the peritoneal spread of tumor cells 22 days following injection.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by Fox Chase Cancer Center’s Institutional Animal Care and Use Committee.
Ovarian cancer is the leading cause of death among all gynecologic malignancies 1. The high mortality rate of this disease is largely due to its late diagnosis and the lack of reliable diagnostic methods 2. Furthermore, conventional chemotherapy often encounters chemoresistance and relapse of cancer 3. Therefore, novel therapeutics is required to effectively target this disease. In development of new therapy targeting human ovarian cancer, it is critical to develop a representative animal model.
An orthotopic animal model has advantages over conventional xenograft models (e.g. subcutaneous or intraperitoneal injections of tumor cells) in that 1) it reproduces the primary site of tumor formation, 2) it represents common site of metastases, and 3) it provides tumor cells to interact with appropriate microenvironment 4, 5, 6, 7, 8. Rodents have a unique bursal membrane that surrounds the ovary and is continuous with the oviduct. This unique anatomy of rodents allows injection of ovarian tumor cells orthotopically. Intrabursally injected ovarian tumor cells behave similar to human disease, thereby growing within intrabursal membrane and spreading into peritoneal cavity as tumor progresses. Injection of cells stably expressing luciferases or fluorescent proteins also allows tracking behavior of tumor cells in real time. Bioluminescent and/or fluorescent imaging technology makes it possible to repeatedly image tumor cells over an extended period of time and study tumor growth, distribution, and regression in non-invasive manner 9, 10, 11.
In establishing the orthotopic model, it is critical to quickly remove the needle from the bursa upon injection. Abrupt removal of the needle seals the puncture site and prevents the leakage of injected cells. However, at the same time, the movement should be gentle not to tear the bursa. If leakage occurs during injection, it can cause cells to seed in the abdomen and potentially confound the study, i.e., premature spreading outside the ovary. In this event, the specific animal should be recorded and followed for the development of multiple tumor sites in the peritoneum prior to treatment or alternatively eliminated from the subsequent analyses. Prior to performing oral gavage, it is critical to check the length of the gavage tube by measuring from the tip of the animal’s head to the last rib. It is helpful to mark the tube at the animal’s nose and do not pass the tube past this mark. Insertion of the tube past this mark can result in a perforation to the stomach. Determining the length of the tube is especially important with younger animals or animals weighing under 20 g. Upon insertion of the gavage needle down the esophagus, there should be no resistance or struggle from the animal. It is important not to administer the solution or suspension too fast as this can lead to reflux and subsequently deliver inaccurate dose volume, as well as adding stress to the animal. For comparable imaging results from time to time, it is desirable to keep the time consistent between injection of luciferase substrate and imaging. The time lapse can be determined by taking series of images after substrate injection and observe the kinetics of signals. Development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging are necessary techniques for better understanding of development and spread of ovarian cancer and assessing novel therapeutic regimens that may ultimately improve the outcome of patient with this deadly disease.
The authors have nothing to disclose.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Ketamine Hydrochloride | Vedco, Inc. | Not Available | ||
Xylazine Hydrochloride | Lloyd, Inc. | Not Available | ||
Acepromazine | Vedco, Inc. | Not Available | ||
D-Luciferin Potassium Salt | Caliper Life Sciences | 122796 |