We thank Dr. Harvey Hensley for his expert technical support.  This work benefited from the use of the following Fox Chase Cancer Center (FCCC) facilities: Laboratory of Animal Facility and Small Animal Imaging Facility.  This work was supported in part by the Ovarian Cancer SPORE at FCCC/UPenn (P50 CA083638), the FCCC core grant (P30 CA006927), and the Ovarian Cancer Research Fund.

I. Preparation of Ovarian Tumor Cells
<ol>
<li>	Grow ovarian cancer cell lines expressing luciferase in culture. Sources of luciferase can be Firefly, Renilla, or other species. Cells expressing fluorescent proteins can also be used. For this demonstration we use an ovarian cancer cell line, OVCAR5, expressing firefly luciferase.</li>
<li>	Harvest cells using routine cell culture technique.</li>
<li>	Keep the cell suspension (10,000 cells/&mu;L) in phosphate buffered saline (PBS) on ice until time of injection.</li>
</ol>

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.
<ol>
<li>	Prepare the anesthetic solution by mixing 3 mL of ketamine hydrochloride (100 mg/mL), 1.6 mL of xylazine hydrochloride (100 mg/mL), 1.5 mL of acepromazine (10 mg/mL), and 20 mL of 0.9% sodium chloride.</li>
<li>	Check the animal's identification number and observable health. Anesthetize the animal with prepared ketamine-xylazine-acepromazine anesthesia via intraperitoneal (i.p.) injection with a dose volume of 8~9 mL/kg body weight (BW).</li>
<li>	Confirm that the animal is under an acceptable plain of anesthesia by performing a toe pinch with forceps or fingers to the animal's hind paws. If there is pedal reflex, wait for a deeper plain of anesthesia until the animal is unresponsive to this procedure.</li>
<li>	Lay the animal dorsal side up on a sterile gauze pad with its head facing away and its tail facing towards you. The point of incision is located to the left or right of the midline and above the ovaries. Shave or wet the fur with 70% alcohol at the incision point.</li>
<li>	Lift the wetted skin using forceps and make a small incision with the scissor at the dorsomedial position and directly above the ovarian fat pad. The ovarian fat pad should be visible beneath the surface of the peritoneal wall. Fat pad is easily recognizable by its white color in contrast to the dark pink tissue surrounding it.</li>
<li>	Gently lift the peritoneal wall lining and make a small incision as described above (5).</li>
<li>	Place a sterile soaked saline gauze pad on the midline adjacent to the incision. Locate the ovarian fat pad and gently pull it out and rest it onto the gauze. Stabilize the ovary by clamping the fat pad with a bulldog clip. Under a dissecting microscope, position the ovary as to allow for the insertion of the needle (30 gauge, G) into the oviduct tubule bend leading to the bursa. When the needle is inserted into the proper position, it should be visible under the bursa.</li>
<li>	Gently push the plunger of the syringe to inject 5 &mu;L of cell suspension between the bursa and the ovary while the syringe is positioned to injection site. This step requires two people. One person pushes the plunger while the other person maintains positioning of needle. Remove the needle quickly to seal the puncture site but gently enough not to tear the bursa and tubule. The bursa should appear to be slightly distended with proper injection.</li>
<li>	Release the fat pad from the bulldog clip and gently replace the reproductive tract and fat pad back into the peritoneal cavity. Gently close the body wall by pulling the upper peritoneal lining over the lower lining. Close the skin with surgical staples or wound clips.</li>
<li>	Place the recovering animal back in its cage and provide a safe heat source to avoid hypothermia and speed up recovery. Monitor the breathing rate and ease, the return of muscle tone, and the ability to voluntarily move. These are all good indicators of the progression towards recovery. Staples or wound clips can be removed 7 or more days post surgery.</li>
</ol>

III. Oral Gavage Administration
<ol>
<li>	Use an 18~20 G gavage needle or feeding tube with a rounded tip. The gavage needle should be no longer than the distance from the tip of the animal's head to its last rib.</li>
<li>	Check the animals' identification number and observable health. Gently scruff the animal by grasping the skin over the shoulders with thumb and fingers. The restraint should be just firm enough for the fore limbs to be extended to each side and out of the way. The animal should not be able to grasp at the needle.</li>
<li>	Gently pull back the animal's head with your index finger, forming a straight line through the neck and esophagus. Support the animal's back with the inside of your thumb.</li>
<li>	Gently and without force, insert the gavage needle at either side of the mouth and over the tongue. In one smooth motion, the needle should pass against the roof of the mouth and down the esophagus. Gravity should guide the needle down without meeting resistance. If there is any resistance, remove the needle and start again. The animal's gag and swallow reflex may be triggered during insertion. However, there should be no resistance or gasping from the animal. It is important to make sure that the animal is breathing when the needle is in place by checking the movement of the nostrils and chest.</li>
<li>	Slowly push plunger of the syringe to dispense the dose volume.</li>
<li>	Gently remove the gavage needle at the same angle and pathway as the way it was inserted down the esophagus.</li>
<li>	Return the animal to its cage and monitor breathing and behavior for 5~10 minutes.</li>
</ol>

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.
<ol>
<li>	Prepare the solution of D-luciferin (substrate of firefly luciferase) by dissolving 5~20 mg in 1 mL PBS and filtering through 0.22 &mu;m membrane for sterilization.</li>
<li>	Charge the induction chamber by turning on both the oxygen and the isoflurane gas. Turn ON the gas flow to the induction chamber only. The isoflurane flow rate is maintained at a low level until animals are ready to be imaged.</li>
<li>	Check the animals' identification number and observable health. Place the animal in the induction chamber charged with isoflurane gas.</li>
<li>	Remove the animal from the chamber when the animal appears to be anesthetized. Deliver 200 &mu;L of prepared luciferin solution via i.p. injection using 30 G needle. It is important to record the time of injection in order to keep the time consistent between luciferin injection and imaging performance throughout the study.</li>
<li>	Place the luciferin-injected animal back into the induction chamber.</li>
<li>	Set the imaging system to the appropriate settings. Be sure to initialize the system before acquiring the first animal image.</li>
<li>	Turn OFF the gas flow to the induction chamber and turn ON the gas flow to the imaging chamber.</li>
<li>	Place the anesthetized animal dorsal or ventral side up. Be sure to place the animals' nose in a nose cone to maintain proper anesthesia induction. Make sure the animal is within the "point of interest grid" as indicated by green illumination.</li>
<li>	Close the door of the chamber and acquire the image at the appropriate time. Time can be determined by kinetics.</li>
<li>	Remove the animal from the imaging chamber and place it back into its cage. Monitor the animals' recovery as described in "Intrabursal Injection".</li>
</ol>

V. Representative Results
<img src="/files/ftp_upload/2125/2125fig1.jpg" alt="Figure 1" /> 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.

<ol>
	<li>Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 59, 225-249 (2009).</li><li>Lynch, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+ovarian+carcinoma:+heterogeneity,+molecular+genetics,+pathology,+and+management.">Hereditary ovarian carcinoma: heterogeneity, molecular genetics, pathology, and management.</a> Mol Oncol. 3, 97-137 (2009).</li><li>Yap, T. A., Carden, C. P., Kaye, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+chemotherapy:+targeted+therapies+in+ovarian+cancer.">Beyond chemotherapy: targeted therapies in ovarian cancer.</a> Nat Rev Cancer. 9, 167-181 (2009).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Metastasis Rev. 17, 279-284 (1998).</li><li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation:+advantages+and+disadvantages.">Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages.</a> Eur J Cancer. 40, 852-857 (2004).</li><li>Shaw, T. J., Senterman, M. K., Dawson, K., Crane, C. A., Vanderhyden, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intraperitoneal,+orthotopic,+and+metastatic+xenograft+models+of+human+ovarian+cancer.">Characterization of intraperitoneal, orthotopic, and metastatic xenograft models of human ovarian cancer.</a> Mol Ther. 10, 1032-1042 (2004).</li><li>Greenaway, J., Moorehead, R., Shaw, P., Petrik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-stromal+interaction+increases+cell+proliferation,+survival+and+tumorigenicity+in+a+mouse+model+of+human+epithelial+ovarian+cancer.">Epithelial-stromal interaction increases cell proliferation, survival and tumorigenicity in a mouse model of human epithelial ovarian cancer.</a> Gynecol Oncol. 108, 385-394 (2008).</li><li>Connolly, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ovarian+cancer.">Animal models of ovarian cancer.</a> Cancer Treat Res. 149, 353-391 (2009).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging.">Bioluminescence imaging.</a> Proc Am Thorac Soc. 2, 537-540 (2005).</li><li>Lehmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+and+multimodal+in+vivo+imaging+of+tumor+hypoxia+and+its+downstream+molecular+events.">Longitudinal and multimodal in vivo imaging of tumor hypoxia and its downstream molecular events.</a> Proc Natl Acad Sci U S A. 106, 14004-14009 (2009).</li><li>Connolly, D. C., Hensley, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+and+Transgenic+Mouse+Models+of+Epithelial+Ovarian+Cancer+and+Non-Invasive+Imaging+Modalities+to+Monitor+Ovarian+Tumor+Growth+In+Situ:+Applications+in+Evaluating+Novel+Therapeutic+Agents.">Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non-Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In Situ: Applications in Evaluating Novel Therapeutic Agents.</a> Current Protocols in Pharmacoogy. 45, (2009).</li></ol>

No conflicts of interest declared.

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.

<table><tbody><tr><td>Ketamine Hydrochloride</td><td>Vedco, Inc.</td><td>Not Available</td><td></td></tr><tr><td>Xylazine Hydrochloride</td><td>Lloyd, Inc.</td><td>Not Available</td><td></td></tr><tr><td>Acepromazine</td><td>Vedco, Inc.</td><td>Not Available</td><td></td></tr><tr><td>D-Luciferin Potassium Salt</td><td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Substrates" target="_blank"><img src="/img/new_window.png" /> Caliper Life Sciences</a></td><td>122796</td><td></td></tr></tbody></table>

in vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer

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.

Watch this Scientific Journal Video about In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer at JoVE.com

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

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.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

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Biology

33.3K Views.  Women's Cancer Program.  中文 

Video: In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

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.

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.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Cancer Research

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Immunology and Infection

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Medicine

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

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 ...

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 ...

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.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

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