Name: Fluorescent Orthotopic Mouse Model of Pancreatic Cancer (Video) | JoVE Journal
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 6 min 48 s
Description: Learn to develop a fluorescent orthotopic mouse model of pancreatic cancer using GFP-expressing PANC-1 cells in Balb-c nude mice. This technique enables non-invasive real-time monitoring of tumor progression and metastasis for drug testing.

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5 minutes

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>Siegel, R. Pancreatic Cancer Stats: American Cancer Society: Cancer Facts & Figures. , Available from:
 <a target="_blank" href="http://www.cancer.org/acs/groups/content/@research/documents/webcontent/acspc-042151.pdf">http://www.cancer.org/acs/groups/content/@research/documents/webcontent/acspc-042151.pdf</a> (2014).</li>
<li>Smyth, E., Cunningham, D. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSmyth%2C+E+%22Harrison%27s+Principles+of+Internal+Medicine%22">Harrison's Principles of Internal Medicine</a>. Kasper, D., et al. , 19th edn, McGraw-Hill Education. (2015).</li>
<li>Mahipal, A., Frakes, J., Hoffe, S., Kim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+borderline+resectable+pancreatic+cancer%5BTitle%5D)+AND+%22World+J+Gastrointest+Oncol%22%5BJournal%5D)">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/pubmed?term=((Diagnosis+and+management+of+pancreatic+cancer%5BTitle%5D)+AND+%22Am+Fam+Physician%22%5BJournal%5D)">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/pubmed?term=((Maximizing+mouse+cancer+models%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">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/pubmed?term=((Patient-derived+orthotopic+xenografts%3A+better+mimic+of+metastasis+than+subcutaneous+xenografts%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">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/pubmed?term=((The+multiple+uses+of+fluorescent+proteins+to+visualize+cancer+in+vivo%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">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/pubmed?term=((Establishment+of+an+orthotopic+pancreatic+cancer+mouse+model%3A+cells+suspended+and+injected+in+Matrigel%5BTitle%5D)+AND+%22World+J+Gastroenterol%22%5BJournal%5D)">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/pubmed?term=((Advances+in+optical+imaging+for+pharmacological+studies%5BTitle%5D)+AND+%22Front+Pharmacol%22%5BJournal%5D)">Advances in optical imaging for pharmacological studies.</a> Front Pharmacol. 6, 189(2015).</li>
<li>Corning. Corning Matrigel Matrix: Frequently Asked Questions. , Available from:
 <a target="_blank" href="http://csmedia2.corning.com/LifeSciences/media/pdf/faq_DL_026_Corning_Matrigel_Matrix.pdf">http://csmedia2.corning.com/LifeSciences/media/pdf/faq_DL_026_Corning_Matrigel_Matrix.pdf</a> (2013).</li>
<li>Metildi, C. A., Kaushal, S., Hoffman, R. M., Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+serial+selection+of+human+pancreatic+cancer+cells+in+orthotopic+mouse+models+produces+high+metastatic+variants+irrespective+of+Kras+status%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">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/pubmed?term=((Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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/pubmed?term=((Survival+efficacy+of+adjuvant+cytosine-analogue+CS-682+in+a+fluorescent+orthotopic+model+of+human+pancreatic+cancer%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Real-time+optical+imaging+of+primary+tumor+growth+and+multiple+metastatic+events+in+a+pancreatic+cancer+orthotopic+model%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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><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



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...

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

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.

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

This video demonstrates the protocol for implanting green fluorescent protein-expressing pancreatic cancer cells orthotopically into the pancreas of the mouse. The orthotopic mouse model helps in investigating the efficacy of novel therapeutics on primary and metastatic tumors.

Fluorescent Orthotopic Mouse Model: Implanting GFP Expressing Cancer Cells into Mouse for Assessing Tumor Progression In Vivo

This video demonstrates an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and in vivo 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.

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

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.

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

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 of an Orthotopic Pancreatic Cancer Mouse Model

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.

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

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.

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

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.

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

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

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

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.

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

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.

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

Learn to develop a fluorescent orthotopic mouse model of pancreatic cancer using GFP-expressing PANC-1 cells in Balb-c nude mice. This technique enables non-invasive real-time monitoring of tumor progression and metastasis for drug testing.

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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