We thank the Western University of Health Sciences for the Intramural Grant.

The protocol described below is executed under guidance and approval of Western University&#39;s Animal Care and Use Committee. All experiments are performed in compliance with all relevant guidelines, regulation and regulatory agencies.
1. Cell Culture
<ol>
	<li>Preparation of Complete Medium
	<ol>
		<li>Using a Class II biological safety cabinet, prepare complete medium by aseptically adding fetal bovine serum (FBS) and penicillin streptomycin (P/S) to a 500 ml bottle of R...

<ol>
	<li>Smyth, E., Cunningham, D., Kasper, D., 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=.">.</a> Harrison's Principles of Internal Medicine. , (2015).</li><li>Mahipal, A., Frakes, J., Hoffe, S., Kim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+borderline+resectable+pancreatic+cancer.">Management of borderline resectable pancreatic cancer.</a> World J Gastrointest Oncol. 7, 241-249 (2015).</li><li>De La Cruz, M. S., Young, A. P., Ruffin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+pancreatic+cancer.">Diagnosis and management of pancreatic cancer.</a> Am Fam Physician. 89, 626-632 (2014).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nat Rev Cancer. 7, 645-658 (2007).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+orthotopic+xenografts:+better+mimic+of+metastasis+than+subcutaneous+xenografts.">Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts.</a> Nat Rev Cancer. 15, 451-452 (2015).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+uses+of+fluorescent+proteins+to+visualize+cancer+in+vivo.">The multiple uses of fluorescent proteins to visualize cancer in vivo.</a> Nat Rev Cancer. 5, 796-806 (2005).</li><li>Jiang, Y. J. <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+an+orthotopic+pancreatic+cancer+mouse+model:+cells+suspended+and+injected+in+Matrigel.">Establishment of an orthotopic pancreatic cancer mouse model: cells suspended and injected in Matrigel.</a> World J Gastroenterol. 20, 9476-9485 (2014).</li><li>Arranz, A., Ripoll, J. <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+optical+imaging+for+pharmacological+studies.">Advances in optical imaging for pharmacological studies.</a> Front Pharmacol. 6, 189 (2015).</li><li>Metildi, C. A., Kaushal, S., Hoffman, R. M., Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+serial+selection+of+human+pancreatic+cancer+cells+in+orthotopic+mouse+models+produces+high+metastatic+variants+irrespective+of+Kras+status.">In vivo serial selection of human pancreatic cancer cells in orthotopic mouse models produces high metastatic variants irrespective of Kras status.</a> J Surg Res. 184, 290-298 (2013).</li><li>Kim, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nat Protoc. 4, 1670-1680 (2009).</li><li>Katz, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+efficacy+of+adjuvant+cytosine-analogue+CS-682+in+a+fluorescent+orthotopic+model+of+human+pancreatic+cancer.">Survival efficacy of adjuvant cytosine-analogue CS-682 in a fluorescent orthotopic model of human pancreatic cancer.</a> Cancer Res. 64, 1828-1833 (2004).</li><li>Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optical+imaging+of+primary+tumor+growth+and+multiple+metastatic+events+in+a+pancreatic+cancer+orthotopic+model.">Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model.</a> Cancer Res. 62, 1534-1540 (2002).</li></ol>

The authors have nothing to disclose.

We describe an orthotopic murine model of pancreatic cancer which expresses GFP, thus allowing non-invasive monitoring of tumor growth using whole body in vivo fluorescent imaging (Figure 1). This technique allows us to monitor the tumor development in real time (Figure 3); it can be an important tool for researchers to study the therapeutic efficacy of novel agents against pancreatic cancer. Another important aspect of this model is that GFP fluorescence provides a visual cue i...

Pancreatic cancer is diagnosed with increased frequency compared to other cancers and is the 4th leading cause of cancer-related deaths in the United States. From the time of diagnosis, over 90% of patients die within five years 1,2. Currently, surgical tumor removal is the only cure for pancreatic cancer, but less than 20% of patients are eligible to undergo surgery mainly because at the time of diagnosis the disease is at an advanced stage and has metastasized 3,4. The lack of specific symptoms makes pancreatic cancer a silent disease; some of the symptoms include abdominal pain, back pain, loss of appetite, jaundice and nausea; which can be easily interpreted as common digestive illnesses 4. For this reason, it is important to develop new pharmacological tools to aid in the diagnosis and treatment of pancreatic cancer.
The use of animal models allows us to understand the biology of pancreatic cancer and provides an insight into applying this knowledge to humans. Xenograft orthotopic models of pancreatic cancer are realistic, because tumors grow in the organ of origin 5. In contrast to heterotopic models, where cell lines or tumor fragments are implanted subcutaneously, orthotopic modeling allows for the recreation of the tumor microenvironment and mimics the interaction of tumor cells with its surroundings 6. The xenograft model described here derives tumors from the human pancreatic cancer cell line PANC-1 GFP, which is genetically engineered to express the green fluorescent protein (GFP). GFP detection enables for a non-invasive imaging and monitoring of tumor growth and metastasis 7. Tumor development occurs rapidly, spontaneously, and closely resembles that of primary tumors of human pancreatic cancer patients 8. Orthotopic models provide a more accurate prediction of drug efficacy in response to therapeutic agents, while mimicking the tumor microenvironment.
As mentioned above, this animal model allows fluorescent detection of tumor growth and metastasis in real time. Fluorescent detection allows for a more direct/live imaging compared to luminescence. With fluorescence the emitted light is a result of an excitation by another light of a shorter wavelength; whereas in luminescence, the emitted light is the result of a chemical reaction and may not have strong emission9. Furthermore, whole body in vivo fluorescent imaging is not detrimental to the animal and allows researchers to monitor tumor growth over time in response to therapeutic treatments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI media 1640&#160;</td>
			<td>Caisson Labs&#160;</td>
			<td>RPL03-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco</td>
			<td>10437-077</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;&#160;</td>
			<td>Thermo Ficher Sci</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel HC&#160;</td>
			<td>Corning&#160;</td>
			<td>354248</td>
			<td></td>
		</tr>
		<tr>
			<td>SutureVet PGA 6-0 PGA</td>
			<td>Henry Schein</td>
			<td>39010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcare or Foamed Antiseptic Handrub</td>
			<td>Steris</td>
			<td>639680</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dubelcco&#39;s Phosphate-Buffered saline)&#160;</td>
			<td>Thermo Ficher Sci</td>
			<td>21300025</td>
			<td></td>
		</tr>
		<tr>
			<td>TB Syringe 27G1/2</td>
			<td>Becton Dickinson</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Blutler Schein</td>
			<td>50562</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketoprofen&#160;</td>
			<td>Fort Dodge Animal Health&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors, 5.5&#34;straight mayo&#160;</td>
			<td>Henry Schein</td>
			<td>22-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1 GFP cell line&#160;</td>
			<td>Anticancer, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Imaging System:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iBOx Scientia, UVP :</td>
			<td>UVP, LLC&#160; Upland, CA.&#160;</td>
			<td colspan="2">Small Animal Imaging System to observe the fluorescent tumor in live animals</td>
		</tr>
	</tbody>
</table>

fluorescent orthotopic mouse model of pancreatic cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

This method describes a surgical orthotopic implantation of fluorescent human pancreatic cancer cells, focusing on the preparation of the cell suspension for injection, proper anesthesia for rodents, delivery of cell suspension via laparotomy, and the use of fluorescent in vivo small animal imaging. The detection of a green fluorescence signal (GFP signal) between two and three weeks post-implantation, provides researchers a visual cue to confirm the presence of a developing panc...

Watch this Scientific Journal Video about Fluorescent Orthotopic Mouse Model of Pancreatic Cancer at JoVE.com

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

The overall goal of this procedure is to develop a mouse orthotopic model of fluorescent pancreatic cancer that can be monitored non-invasively in a live animal. This model allows a non-invasive study of the effects of a specific drug of interest on a primary tumor and its metastasis in a live animal. The main advantage of this technique is that the effects of the treatments can be easily observed in situ.Begin by thawing an aliquot of high-concentration Matrigel according to the manufacturer's instructions. Next, wash each flask of cells with five milliliters of DPBS, and harvest the cultures with trypsin. After adding RPMI medium, transfer the resulting cell suspensions into 15-milliliter tubes and spin down the cells.At the end of the centrifugation, transfer the cells to a bio-safety cabinet and re-suspend the pellets in 10 milliliters of fresh DPBS with gentle pipetting. After counting, centrifuge the cells again and dilute the cell suspensions to a concentration of three times 10 to the sixth cells per 50 microliters of ice-cold, serum-free medium and Matrigel, a high-concentration, basement-matrix membrane. Then, gently vortex the samples and place them on ice.To implant the cells, first apply ointment to an anesthetized mouse's eyes and place the animal on a heating pad covered with a sterile drape. Then, gently disinfect the flank of the animal with three iodine scrubs, followed by three 70%ethanol rinses. After confirming a lack of response to toe pinch, load approximately 200 microliters of the prepared cells into a pre-cooled one-milliliter TB syringe equipped with an 18-gauge needle.Replace the 18-gauge needle with a 27-gauge needle and place the cells back on ice. Then, locate the general area of the spleen in the upper left quadrant of the abdomen. Using forceps, pinch the skin above the spleen and make an approximately one-centimeter incision to create a pocket.Then, pinch the smooth muscle on top of the spleen and cut through the tissue to access the peritoneal cavity. Next, gently grab the caudal end of the spleen and pull it out of the body cavity. Using a wet, sterile, cotton swab, spread the pancreas attached to the end of the spleen to locate the pancreatic tail.Deliver 50 microliters of the cells into the pancreatic tail, then slowly rotate the needle out of the pancreas. A successful implantation will look like a superficial bubble without any leaks. Return the organs to the peritoneal cavity and enclose everything with the muscle and skin.Then, use a 6-0 suture to close the incision. Afterwards, inject the animals with ketoprofen and monitor them until they are fully recovered. At the appropriate experimental time point, use a commercial small-animal image system to image the tumor growth in the live animal under anesthesia and select the appropriate excitation and emission filters.Next, obtain an initial image using white light only. Keeping the animal in the same position, switch to the GFP filters and acquire a second image. To analyze the tumor growth, superimpose the white and fluorescent images to assess the fluorescent area and intensity of the tumor cells.The detection of a green fluorescent signal between two and three weeks post-implantation provides a visual cue for confirming the presence of a developing pancreatic cancer tumor, as the animals that do not develop tumors do not exhibit a GFP signal. Further, this method allows non-invasive monitoring of the tumor growth progression, with an increase in the GFP signal observed over time as the tumor size increases. Metastasis of the tumor to specific organs can be confirmed upon removal of the tissues of interest for further ex vivo fluorescent imaging.Once mastered, this technique can be completed in six to eight minutes and can be used to study the effects of different chemotherapeutic agents on pancreatic cancer and its metastasis. While attempting this procedure, it's important to keep the Matrigel in ice, or else the gel will be hard to inject. Also, it is important to remember to use aseptic techniques and to monitor all the anesthesia levels at all times.After watching this video, you should have a good understanding on how to produce an orthotopic model of pancreatic cancer in mice. Don't forget that working with human cancer cell lines can be hazardous, and that precautions such as wearing protective equipment and properly disposing biological waste, should be taken at all times.

fluorescent-orthotopic-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

16.3K Views.  Western University of Health Sciences. The overall goal of this procedure is to develop a mouse orthotopic model of fluorescent pancreatic cancer that can be monitored non-invasively in a live animal. This model allows a non-invasive study of the effects of a specific drug of interest on a primary tumor and its metastasis in a live animal. The main advantage of this technique is that the effects of the treatments can be easily observed in situ.Begin by thawing an aliquot of high-concentration Matrigel according to the manuf...