Name: fluorescent orthotopic mouse model of pancreatic cancer
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Fluorescent Orthotopic Mouse Model of Pancreatic Cancer at JoVE.com

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 RPMI medium. Mix gently by swirling. The final concentration of each supplement is 10% FBS and 1% P/S (v/v). For example, supplement 500 ml of media with 56 ml FBS and 6 ml P/S.</li>
		<li>Place complete medium in a water bath at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Thawing and Propagating Cells
	<ol>
		<li>Retrieve PANC-1 GFP cells from liquid nitrogen. Thaw the vial by gentle agitation in a water bath at 37 &#176;C. 
		Note: Thawing should be rapid (approximately 2 - 3 min).</li>
		<li>Label T-75 flasks to be used with (a) cell-line name, (b) passage number, (c) date, and (d) researcher&#39;s initials.</li>
		<li>Wipe the vial with 70% ethanol and transfer vial to a biosafety cabinet.</li>
		<li>To propagate the cells, add 9.0 ml of complete medium to a 15 ml conical tube. Using a 1.0 ml pipette, carefully transfer the cell suspension and suspend it in 9 ml of complete medium.</li>
		<li>Centrifuge at 125 x g for 8 min at RT. Transfer the conical vial to a biosafety cabinet and aspirate the supernatant without disturbing the pellet.</li>
		<li>Suspend the pellet in 10 ml of fresh complete medium by gently pipetting the suspension up and down.</li>
		<li>Count the cells with a hemocytometer and plate them in T-75 flasks at a density of 2.1 x 106 cells/flask. Place flask in an incubator at 37 &#176;C and 5% CO2.</li>
		<li>Monitor the cells daily by eye and under the microscope. If fluid renewal is needed, aseptically aspirate the complete growth medium from the flask and discard. Add equal volume of fresh complete medium.</li>
	</ol>
	</li>
	<li>Propagating Cells
	<ol>
		<li>Aseptically remove medium from the flask. Add 5 ml of Dulbecco&#39;s phosphate buffer saline (DPBS) to rinse cells and discard.</li>
		<li>Detach the cells from the flask by adding 3 ml of 0.25% trypsin. Incubate flask containing trypsin at 37 &#176;C and 5% CO2 for 5 to 10 min.</li>
		<li>Observe cells under the microscope (10X magnification) and ensure they are round and dislodged.</li>
		<li>Neutralize trypsin by adding an equal volume of complete medium and break up clumps by gently pipetting up and down.</li>
		<li>Count the cells using a hemocytometer and transfer the appropriate cell suspension volume to new flasks at the cell density mentioned in 1.2.7; add enough fresh media so the final volume is 10 ml per flask.</li>
	</ol>
	</li>
	<li>Cell Suspension Preparation for Injection 
	Note: Since the basement membrane matrix, high concentration, solidifies at RT, keep all materials on ice. Place all sterile pipette tips and vials in the freezer overnight, and keep on ice while working on the suspension.
	<ol>
		<li>Keep a bottle of RPMI media without any additives on ice. Thaw the basement membrane matrix, high concentration, by following manufacturer&#39;s instructions. Preferably, aliquot into smaller vials to avoid multiple freeze-thaw cycles 10.</li>
		<li>Aspirate media from all flasks, and rinse with 5 ml of DPBS. Repeat all steps on the previous section up to step 1.3.4.</li>
		<li>Transfer contents to a 15 ml conical tube and centrifuge at 125 x g for 8 min at RT. Transfer conical vial to biosafety cabinet and re-suspend pellet using 10 ml of DPBS.</li>
		<li>Gently pipette the suspension up and down to break up the pellet. Count cells using a hemocytometer.</li>
		<li>Centrifuge again as outlined in step 1.4.3. Aspirate supernatant and depending on the number of cells, add appropriate diluent to yield 3 x 106 cells into a volume of 50 &#181;l. The diluent will be a mixture of serum free media and high concentration basement matrix membrane in a 1:1 (v/v) ratio.</li>
		<li>Vortex the suspension gently and keep on ice at all times. The suspension is now ready for injection.</li>
	</ol>
	</li>
	<li>Surgical Implantation 
	Note: Ensure that all surgical materials and instruments are sterile. Practice aseptic techniques at all times.
	<ol>
		<li>Place a heating pad on the table and cover with a sterile drape. Set up the anesthesia machine and ensure all the supplies are within arm&#39;s reach.</li>
		<li>Place the animal&#39;s snout into the anesthesia mask and set up the anesthesia to 1 L/min of oxygen and 2.5% isoflurane.</li>
		<li>Gently scrub the flank of the animal with iodine, and rinse with 70% ethanol. Repeat three times.</li>
		<li>Confirm the animal is fully anesthetized by pinching the hind paw. The animal is fully anesthetized and ready for surgery if no response is observed. However, if the animal flinches, ensure there is sufficient anesthesia in the vaporizer and allow the animal more time to go completely under anesthesia.</li>
		<li>Load approximately 200 &#181;l of the cell suspension into a 1.0 ml TB syringe with an 18 G needle (previously cooled). Replace the needle with a 27 G needle and return to ice until ready to use.</li>
		<li>Locate the general area of the spleen (left upper quadrant of the abdomen) and using forceps pinch the skin on top of that region. Using surgical scissors make an incision of approximately 1.0 cm to create a pocket. Similarly, pinch the smooth muscle on top of the spleen and cut through in order to access the peritoneal cavity.</li>
		<li>Gently grab the caudal end of the spleen and pull it out of the body. The pancreas will be attached to the spleen. Spread the pancreas using a wet sterile Q-tip and locate the tail of the pancreas.</li>
		<li>Deliver the 50 &#181;l injection into the tail of pancreas, leave the needle inside for 10 sec and slowly rotate the needle out of the pancreas. A successful implantation will look like a superficial bubble without any leaks.</li>
		<li>Return the pancreas and spleen to the peritoneal cavity. First enclose the muscle and then enclose the skin separately. Use a 6-0 suture or staple to close the incision.&#160;To avoid pain in the animal, administer ketoprofen subcutaneously (SC) (5 mg/kg) over 24 hr. Alternatively, administer buprenorphine sc (0.05 - 0.1 mg/kg) every 12 hr over a 36 hr period.&#160;&#160;</li>
		<li>Recover the animal from anesthesia and return it to its cage. Also monitor for pain. Animals must be provided with pain relief at the time of (or even prior to &#8212; pre-emptive) surgery. Additional pain relief must be provided to animals that experience pain according to IACCUC-protocol or as prescribed by the attending veterinarian or designee.</li>
	</ol>
	</li>
	<li>In vivo Imaging 
	Note: Image capture was performed using a commercial imaging system equipped with a dark chamber and the appropriate filters for GFP imaging. Image acquisition was accomplished using a CCD camera and an optical system consisting of interchangeable excitation/emission lenses (excitation: 455 - 495 nm; emission: 513 - 557 nm). Bright field images were captured without a filter at 1 x 1 binning for each time point. Excitation of GFP utilized a xenon multispectral light source. Animals were maintained under anesthesia throughout the imaging process by connecting the anesthesia machine to a gas anesthesia manifold integrated into the dark chamber of the imaging system.
	<ol>
		<li>Anesthetize the animal as outlined in 1.5.2. An anesthesia mask is in the interior of the commercial imaging system&#39;s dark chamber in order to keep the animal under anesthesia during the imaging process.</li>
		<li>Set up the correct excitation and emission filters.</li>
		<li>Begin by obtaining an initial image using white light only. Keep the same position of the animal throughout the imaging session and switch to GFP filters to take a second image.</li>
		<li>For best results superimpose both images. Analyze the image for fluorescent area and intensity.</li>
	</ol>
	</li>
</ol>

<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 indicating successful implantation and growth of pancreatic cancer; which would otherwise be difficult to gauge in live animals. Consistent with the clinical setting, this model provides an insight on metastasis. It shows metastasis to the surrounding organs: spleen, mesenteric lymph nodes, liver, and gastrointestinal tract (Figure 4). We observed metastases as early as 7 weeks post transplantation. PANC-1 metastases have been reported in the range of 10 - 18 weeks11. The time at which metastasis is observed may depend on the number of implanted cells, implantation and visualization techniques. In the current study, we chose a green fluorescent cell line, PANC-1 GFP, which is commercially available. The model described here is reproducible and the metastases can be easily visualized because they have bright fluorescence. A limitation of xenograft nude mouse models produced from established cancer cell lines is the possibility of reduced tumor heterogeneity compared to an original human tumor12; nevertheless the model is reproducible, easy to develop and follow its progress in live animals. Furthermore, xenograft mice models continue to be used widely in cancer research.
The fluorescently labeled cells must be pelletized and re-suspended in ice cold serum-free medium mixed with equal volume of ice-cold basement membrane 8. The cell suspension must be maintained on ice at all times to prevent gelling or solidification. It is important to load the syringes with an 18 gauge needle in order to avoid lysing the cells. If the cells are lysed, the cell suspension is rendered useless, and no tumor growth will be attained (Figure 2). Immediately post-injection, allow approximately 10 sec for the cell suspension to solidify prior to removing the needle from the injection site. The solidification properties of the suspension allowed us to inject the cells without leakage from the injection site. Leakage of cells can lead to metastasis as an artifact rather than from cell dissemination. We have optimized this orthotopic implantation of PANC-1 GFP cell line using 3 x 106 cells per 50 &#956;l injection; other cell line implantations must be determined empirically. Higher volumes of injection up to 100 &#181;l containing 500,000 cells have been reported in the literature12. The main optimization parameters taken into account were successful orthotopic tumor growth and positive tumor imaging via GFP fluorescence within three weeks post-implantation.
The tumor growth and development is not only affected by the number of cells injected, but also by the age of the mice used. We have used athymic nude mice and determined that using young mice between the ages of 6 to 8 weeks yields more reproducible results when compared to older mice. As these mice age, they begin to regain some immunity which may result in rejection of the human pancreatic cells. The imaging techniques described here are not limited to orthotopic use; they can also be used for heterotopic implantation of tumors. Care must be taken to avoid auto-fluorescence which may obscure the tumor GFP signal. Certain plastics used for anesthesia tubing may produce auto-fluorescent artifacts.
Fluorescent whole-body imaging enables rapid analysis of tumor growth and progression 13. A major advantage of using fluorescence is the ability to track cancer without the traditional cumbersome procedures of histopathological examination or immunohistochemistry 14. This model has bright fluorescence and enables image acquisition without the need of skin flaps or other manipulations. Fluorescence differs from bioluminescence in that it does not create light based on a chemical reaction; it simply absorbs light and reemits it at a lower frequency. Orthotopic xenograft models provide invaluable knowledge and understanding of pancreatic tumor biology which can be translated into novel therapies for human use.

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><tbody><tr><td>RPMI media 1640</td><td>Caisson Labs</td><td>RPL03-500ML</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>10437-077</td><td></td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;&amp;nbsp;</td><td>Thermo Ficher Sci</td><td>15140-122</td><td></td></tr><tr><td>Matrigel HC basement membrane, high concentration</td><td>Corning</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&#039;s Phosphate-Buffered saline)&amp;nbsp;</td><td>Thermo Ficher Sci</td><td>21300025</td><td></td></tr><tr><td>TB Syringe 27 G 1/2</td><td>Becton Dickinson</td><td>305620</td><td></td></tr><tr><td>Isoflurane</td><td>Blutler Schein</td><td>50562</td><td></td></tr><tr><td>Ketoprofen</td><td>Fort Dodge Animal Health</td><td></td><td></td></tr><tr><td>Surgical Scissors, 5.5&quot; straight mayo&amp;nbsp;</td><td>Henry Schein</td><td>22-1600</td><td></td></tr><tr><td>PANC-1 GFP cell line&amp;nbsp;</td><td>Anticancer, Inc</td><td></td><td></td></tr><tr><td>Small Animal Imaging System: iBox Scientia</td><td>UVP, LLC. Upland, CA.</td><td></td><td>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 pancreatic cancer tumor (Figure 1). Figure 1 consists of three images of a mouse with PANC-1 GFP fluorescent tumor. The first is taken under white light (Figure 1A); the second image is taken under blue fluorescent light (excitation: 455 - 495 nm; emission: 513 - 557 nm) to image the green fluorescence emitted from the PANC-1 pancreatic tumor (Figure 1B); the third is a composite of the first two and shows the location of the tumor within the body of the mouse (Figure 1C). Animals which do not develop tumors do not show GFP signal (Figure 2). Furthermore, representative images of tumor progression over time may be non-invasively monitored, by recording the GFP signal at different time points (Figure 3). Figure 3 shows several composites of GFP signals over time. As the time progresses and the tumor size increases, the GFP signal increases. Twenty days after the implantation, the tumor appears as a small green dot, and 50 days after implantation, the tumor size increases significantly. Figure 4A shows metastases to the spleen, liver, and gastrointestinal tract, which can be confirmed after the animal has been euthanized and the organs are removed for ex vivo fluorescent imaging (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/54337/54337fig1.jpg" /> 
Figure 1: Fluorescent Imaging of Orthotopic Pancreatic Tumor. Balb/c nude mouse was anesthetized with isoflurane and a series of images were obtained using a fluorescent in vivo imaging system: (A) Image was obtained with white light and no filter. (B) Image was obtained using blue light and specific filters for GFP. (C) A composite image of A and B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54337/54337fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54337/54337fig2.jpg" /> 
Figure 2: Implantation Troubleshooting, Lack of Fluorescence. Representation of a mouse at a time where the tumor had not yet developed: (A) Image was obtained with white light and no filter. (B) Image was obtained using blue light and specific filters for GFP. (C) A composite image of A and B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54337/54337fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54337/54337fig3.jpg" /> 
Figure 3: In vivo Real Time Fluorescent Imaging to Track Primary Tumor Growth. In vivo imaging of PANC-1 GFP pancreatic cancer progression at various time points post-implantation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54337/54337fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54337/54337fig4.jpg" /> 
Figure 4: Evidence of Metastasis. (A) A mouse under anesthesia with metastatic tumors; and (B) its primary tumor and metastases ex vivo 100 days post-implantation. Thick arrow shows primary tumor, and thin arrows indicate metastatic tumors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54337/54337fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

16.5K Views.  Western University of Health Sciences. 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. 

Video: Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Medicine

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Adoptive Therapy Using Tumor-Infiltrating Lymphocytes in Pancreatic Cancer Models

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient&#39;s own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration.&#160;Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Bioluminescent Orthotopic Pancreatic Cancer Mouse Model: A Non-invasive Technique to Monitor Cancer Progression in Mice

Source: Chai, M. G., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/50395/" target="_blank">Bioluminescent Orthotopic Model of Pancreatic Cancer Progression</a>.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and&#160;in vivo&#160;bioluminescence imaging for non-invasive monitoring of tumor progression. The orthotopic model is suited to both syngeneic and xenograft models and could be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Source: Chai, M. G., et al. Bioluminescent Orthotopic Model of Pancreatic Cancer Progression.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an ...

JoVE Encyclopedia of Experiments

Pancreatic Cancer

Assays & techniques

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

Source: An, X. et al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57460/" target="_blank">Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry</a>. J. Vis. Exp. (2018)
The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more accurately.

Source: An, X. et al.&#160;Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry. ...

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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