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 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 ...

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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

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

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 ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

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 ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. 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 ...