We would like to thank the TUM animal facility and the imaging core facility of the Department of Nuclear Medicine, Klinikum rechts der Isar, for excellent technical support. This study was supported by the German Cancer Consortium (DKTK), Deutsche Forschungsgemeinschaft (DFG SA 1374/4-2, DFG SA 1374/6-1, SFB 1321 Project-ID 329628492 P06, P11 and S01) to D.S., the Wilhelm Sander-Stiftung (2020.174.1 and 2017.091.2) to D.S., and the European Research Council (ERC CoG No. 648521, to D.S.).

The animal experiments were approved by the institutional animal care and use committees (IACUCs) of the local authorities of Technical University of Munich and Regierung von Oberbayern.
1. Information to consider prior to the procedure
<ol>
	<li>Ensure approval of the animal protocol and personnel by the local authorities before starting any animal experimentation.</li>
	<li>Select recipient mice.
	<ol>
		<li>Select mice of similar ages for implantation (C57Bl/6J recipient mice with an age range between 2 months and 4 months).</li>
		<li>Ensure that the sex and genetic background of the recipient animals match the sex and genetic background of the animals from which the cell line used for implantation originated (syngeneic allografts).</li>
	</ol>
	</li>
	<li>Prepare the mice for implantation. 
	NOTE: Handle the mice according to the hygienic standards of the local animal facility.
	<ol>
		<li>On the day of implantation, do not feed the mice 4 h before the procedure to avoid complications due to the administration of anesthetics.</li>
		<li>Weigh the mice prior to implantation to calculate the amount of the necessary anesthetics.</li>
		<li>Transfer the mice to the operation room.</li>
	</ol>
	</li>
	<li>Select the PDAC cell lines.
	<ol>
		<li>Make sure that the genetic background and sex of the mice from which the cell lines are derived match the background and sex of the recipient mice (e.g., the PDAC cell lines PDAC 1-3 previously generated from PDAC GEMMs on a C57Bl/6J background in the laboratory with the following genotypes, as described previously14: PDAC 1 and 2: Ptf1aCre/+;LSL-KrasG12D/+;LSL-Trp53R172H/+, PDAC 3: Ptf1aCre/+;LSL-KrasG12D/+;LSL-Trp53R172H/R172H).</li>
		<li>Select the cell lines that do not express proteins that are potentially immunogenic.</li>
	</ol>
	</li>
	<li>Culture the PDAC cell lines. 
	NOTE: Perform the cell culture work under a laminar flow hood. Disinfect the gloves, working space, and materials before working.
	<ol>
		<li>Thaw the cells 1 week before implantation by resuspending the pellet in 5 mL of cell culture medium. Spin down the cells, aspirate the supernatant, resuspend the pellet in 5 mL of phosphate-buffered saline (PBS), and spin down the cells again. Remove the supernatant and resuspend the cells in 5 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% fetal bovine serum (FBS) and 0.1% penicillin-streptomycin (PS). Transfer the cell suspension to a 25 cm&#178;&#160;flask.</li>
		<li>Passage the cell lines at least once before implantation.
		<ol>
			<li>Remove the cell medium, wash 1x with PBS, and add 0.5 mL of trypsin to detach the cells from the flask. Incubate the cells at 37 &#176;C until they detach from the flask (depending on the cell line, this usually takes 5-10 min). When the cells are detached, resuspend the cells in 10 mL of DMEM with 10% FBS and 0.1% PS and transfer the cell suspension to a 75 cm&#178; flask. 
			NOTE: As FBS inactivates trypsin, it would hinder trypsinization.</li>
		</ol>
		</li>
		<li>Ensure confluency of ~70%-80% on the day of implantation.</li>
	</ol>
	</li>
	<li>Material needed for implantation
	<ol>
		<li>Autoclave all the surgical instruments.</li>
		<li>Keep anesthetics ready based on the animal experiment license.
		<ol>
			<li>Use meloxicam for analgesia at least 5 min in advance as a long-lasting analgetic with anti-inflammatory properties.</li>
			<li>For surgery, use a combination of midazolam, medetomidine, and fentanyl (MMF) for analgosedation.</li>
			<li>Use a combination of atipamezole, flumazenil, and naloxone (AFN) for antagonizing the MMF analgosedation after implantation.</li>
		</ol>
		</li>
		<li>Prepare a sterile drape, a heating mat, a shaver, gloves, eye cream, 0.9% sodium chloride, iodine or chlorhexidine-based surgical scrub, 80% ethanol, soluble suture, wound clips, a 50 &#181;L injection volume syringe, and tape.</li>
	</ol>
	</li>
</ol>
2. Preparing the cell lines before implantation
NOTE: Prepare the tumor cells only when the mice are ready for implantation. Ensure a short time frame between the harvesting or collection of cells and implantation.
<ol>
	<li>Warm all the liquids to 37 &#176;C before use.</li>
	<li>Prepare the cells (starting from 1x T75 (a 75 cm2 culture flask) as described in step 1.5.2.1 until they detach from the flask. Resuspend the cells in 10 mL of DMEM with 10% FBS and 0.1% PS and transfer the cell suspension to a 15 mL tube.</li>
	<li>Spin down the cells at 200 &#215; g for 5 min in a centrifuge. Aspirate the supernatant and resuspend the cell pellet in an adequate volume of DMEM without additives based on the required final concentration of the cells for injection. Use 10 &#181;L of the cell suspension to count the cells using a Neubauer chamber and calculate the number of available cells.</li>
	<li>Dilute the cells to the final required number of cells for injection (e.g., 2,500 cells in 20 &#181;L) in DMEM without additives. Transfer 1 mL of the diluted cell suspension to a 1.5 mL tube. Place the tube on a rotator until implantation to prevent the cells from aggregating.</li>
</ol>
3. Orthotopic implantation of PDAC cells
NOTE: In case of insufficient surgical experience, practice with cadavers first and get adequate training, for example, within an animal training protocol. The personnel performing the surgery and the animal experiments need to fulfill the criteria of the respective authorities and the institutional guidelines.
<ol>
	<li>Use meloxicam as a perioperative analgesic and apply 5 mg/kg body weight subcutaneously.</li>
	<li>Intraperitoneally inject MMF according to the body weight of each mouse (midazolam [5.0 mg/kg], medetomidin [0.5 mg/kg], and fentanyl [0.05 mg/kg]).</li>
	<li>Put the mouse back into the cage for 10-15 min.</li>
	<li>Apply eye cream when the mouse is asleep.
	<ol>
		<li>Check sufficient analgosedation after 10 min by testing the pedal withdrawal reflex (pinch on the foot pads of both hind feet). In case of a response, apply 1/3 of the original dose of analgosedation. After 10 min, repeat the procedure and proceed only in the absence of a pedal withdrawal reflex.</li>
	</ol>
	</li>
	<li>Shave the left lateral abdominal flank of the mouse. Place the mouse lying on the right side on a sterile heating mat and tape its legs to the surface. Disinfect the abdomen using an iodine or chlorhexidine-based surgical scrub followed by 80% ethanol in a circular motion and repeat the procedure three times. To maintain sterility of the surgical field, use a surgical drape.</li>
	<li>Wear new gloves and disinfect them. Use surgical scissors to cut the skin and subcutaneous fat tissue longitudinally on the left flank where the spleen is projected. Perform a cut of about 1 cm in length.</li>
	<li>Using scissors and forceps, separate the skin from the peritoneum to get an easier access to the peritoneum.</li>
	<li>Change the pair of scissors. Open the peritoneum at the location of the spleen with a 1 cm longitudinal cut.</li>
	<li>Mobilize the spleen and the attached pancreas using blunt, fine-tipped forceps. Make sure the organs are not attached to other tissue and check for supplying blood vessels to avoid injuring the vessels or the surrounding tissue when handling the pancreas. 
	NOTE: Do not grab the spleen directly to prevent bleeding.</li>
	<li>Carefully pull the pancreas out of the incision to enable easier access to the organ&#39;s tail and observe the spleen following the pancreas. Use the non-dominant hand to grab the pancreas with forceps and carefully cut the ligaments that prevent the pancreas from extracorporalization. Make sure the organ is not folded during injection and that the organ&#39;s capsule is held under tension at the site of needle penetration to avoid spillage.</li>
	<li>Use the dominant hand to carry out the injection. Carefully penetrate the organ&#39;s capsule with the syringe&#39;s needle following the longitudinal axis of the organ. Aim for a piece of pancreatic tissue between visible blood vessels to minimize the risk of puncturing or injecting into a vessel. Slowly inject an adequate number of cells according to the experimental plan (here, 2,500 cells in 20 &#181;L of DMEM) with a 27 G cannula and a 50 &#181;L syringe into the tail area of the pancreas. 
	NOTE: A transparent bubble forms upon successful injection into the tissue.</li>
	<li>Keep the pancreas and the inserted syringe still for approximately 1 min to avoid spilling of the tumor cells. Remove the needle carefully from the injection site. 
	NOTE: Change needles between mice and use a new sterile syringe for the injection of a new cell line.</li>
	<li>Rearrange the organs inside the abdomen carefully. Take care not to injure the tissue to avoid spilling of the cells. Pour 1 mL of 0.9% sodium chloride onto the organs to avoid organ adhesion.</li>
	<li>Carefully suture the peritoneum using the simple interrupted suture technique and close the skin using two to three 9 mm stainless steel wound clips. 
	NOTE: Remove the wound clips 7 days after implantation if the surgical incision has healed.</li>
	<li>If necessary, prevent dehydration of the animals due to the procedure and the anesthesia by injecting 0.5 mL of sodium chloride subcutaneously.</li>
	<li>After implantation, antagonize the analgosedation by MMF with a subcutaneous injection of AFN (atipamezol [2.5 mg/kg], flumazenil [0.5 mg/kg], naloxon [1.2 mg/kg]), according to the body weight of the mouse.</li>
	<li>Place the mouse in a 37 &#176;C heated chamber until it is awake and completely active. Then, place the mouse back into its original cage. Monitor the health status of the mouse by checking the fur, skin color, and activity and repeat this 3-4 h after surgery.</li>
</ol>
4. Aftercare for the mice
<ol>
	<li>Give the mice access to water and food when back in the animal facility.</li>
	<li>Continue subcutaneous or oral application of meloxicam (5 mg/kg) every 6-12 h for 2-3 days after surgery.</li>
	<li>Monitor the health status of the mice regularly according to the animal protocol and the institutional guidelines.</li>
	<li>Depending on the goal of the experiment, monitor tumor growth every week or more frequently by palpating, ultrasound imaging, bioluminescence (BLI), or magnetic resonance imaging (MRI). 
	&#8203;NOTE: Based on the method of choice, the growth rate, and the number of cells implanted, a tumor can be detected as early as 1 week after the injection.</li>
</ol>
5. Harvesting tumor grafts
<ol>
	<li>Harvest the tumors at the end of the experiment or when the mice need to be euthanized following the criteria of the institutional animal care and use committees or animal experiment license.</li>
	<li>Euthanize the mouse using a method permitted by the animal experiment license. 
	NOTE: Carry out steps with a minimal time loss to minimize the ischemia time. Therefore, cervical dislocation is recommended as it is a quick method and the time of death can be determined exactly, in contrast to terminal bleeding or chemical methods.</li>
	<li>Place the mouse supine on a sterile mat and tape its legs to the surface. Use 80% ethanol to disinfect the abdomen.</li>
	<li>Wear new gloves and disinfect them. Use surgical scissors to cut the skin longitudinally on the central abdomen.</li>
	<li>Using scissors and forceps, separate the skin from the peritoneum to get an easier access to the peritoneum.</li>
	<li>Change the pair of scissors. Open the peritoneum at the central abdomen with a 5 cm longitudinal cut.</li>
	<li>Mobilize the spleen and the attached pancreas using blunt, fine-tipped forceps. Carefully detach the tumor from the surrounding tissue using forceps and surgical scissors.</li>
	<li>Store the tumor tissue in a medium suitable for downstream experiments. Process the tissues immediately or, if necessary, keep the sample at 4 &#176;C for a maximum of 6 h until further processing.</li>
</ol>

<ol><li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+statistics%2C+2020%5BTitle%5D)+AND+%22CA%3A+A+Cancer+Journal+for+Clinicians%22%5BJournal%5D)">Cancer statistics, 2020.</a> CA: A Cancer Journal for Clinicians. 70 (1), 7-30 (2020).</li>
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<li>Schneider, G., Schmidt-Supprian, M., Rad, R., Saur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-specific+tumorigenesis%3A+Context+matters%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Tissue-specific tumorigenesis: Context matters.</a> Nature Reviews Cancer. 17 (4), 239-253 (2017).</li>
<li>Olive, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+Hedgehog+signaling+enhances+delivery+of+chemotherapy+in+a+mouse+model+of+pancreatic+cancer%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.</a> Science. 324 (5933), 1457-1461 (2009).</li>
<li>Ruscetti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Senescence-induced+vascular+remodeling+creates+therapeutic+vulnerabilities+in+pancreas+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Senescence-induced vascular remodeling creates therapeutic vulnerabilities in pancreas cancer.</a> Cell. 181 (2), 424-441 (2020).</li>
<li>Aung, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomics-driven+precision+medicine+for+advanced+pancreatic+cancer%3A+Early+results+from+the+COMPASS+trial%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">Genomics-driven precision medicine for advanced pancreatic cancer: Early results from the COMPASS trial.</a> Clinical Cancer Research. 24 (6), 1344-1354 (2018).</li>
<li>Chan-Seng-Yue, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcription+phenotypes+of+pancreatic+cancer+are+driven+by+genomic+events+during+tumor+evolution%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution.</a> Nature Genetics. 52 (2), 231-240 (2020).</li>
<li>Kalimuthu, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+classification+of+pancreatic+ductal+adenocarcinoma+that+predicts+molecular+subtypes+and+correlates+with+clinical+outcome%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Morphological classification of pancreatic ductal adenocarcinoma that predicts molecular subtypes and correlates with clinical outcome.</a> Gut. 69 (2), 317-328 (2020).</li>
<li>Hayashi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+unifying+paradigm+for+transcriptional+heterogeneity+and+squamous+features+in+pancreatic+ductal+adenocarcinoma%5BTitle%5D)+AND+%22Nature+Cancer%22%5BJournal%5D)">A unifying paradigm for transcriptional heterogeneity and squamous features in pancreatic ductal adenocarcinoma.</a> Nature Cancer. 1 (1), 59-74 (2020).</li>
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<li>Falcomata, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+multi-kinase+inhibition+sensitizes+mesenchymal+pancreatic+cancer+to+immune+checkpoint+blockade+by+remodeling+the+tumor+microenvironment%5BTitle%5D)+AND+%22Nature+Cancer%22%5BJournal%5D)">Selective multi-kinase inhibition sensitizes mesenchymal pancreatic cancer to immune checkpoint blockade by remodeling the tumor microenvironment.</a> Nature Cancer. 3 (3), 318-336 (2022).</li>
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The authors have no conflicts of interest to disclose.

Syngeneic mouse orthotopic allografts represent a robust model for preclinical studies due to their cost-effectiveness, reproducibility, and relatively simple experimental procedures13,15. These models not only allow the study of tumor-host interactions but also guarantee the preservation of the genetic heterogeneity of the tumors they originate from when primary mouse cell cultures are used for the experiment.
This protocol presents a...

Recently, PDAC became the third leading cause of cancer-related deaths in the western world1. It causes the highest death rate among all cancers and a 10 year overall survival rate of ~1%, which has not changed for decades2. Due to the lack of progress in PDAC treatment, this disease is expected to become the second leading cause of cancer-related deaths by the next decade3.
PDAC tumors are complex entities characterized by a diverse tumor microenvironment (TME) composed of a heterogeneous assembly of stroma, vascular, immune, and extracellular matrix components4. Differences in the composition of the TME influence disease prognosis and response to therapy4,5,6. Indeed, many studies have shown that the basal-like, mesenchymal subtype of PDAC is associated with a highly immunosuppressive TME and shows decreased survival and lack of response to therapies7,8,9,10,11,12. Therefore, a deeper understanding of the differences in TME composition and how these features influence tumor biology remains an important factor for the development of molecularly precise therapies. To better understand the biology behind this complex phenotype and identify therapeutic strategies able to overcome the barrier that the TME of PDAC constitutes, in vivo models are indispensable.
A key aspect for any cancer preclinical model system is that it should mimic human phenotypes, recapitulating both the genetic heterogeneity and the milieu incorporating the multitude of stromal and immune populations that make up the TME. Therefore, when choosing mouse models for preclinical research, several aspects must be taken into consideration. To investigate the tumor-immune interaction, histocompatible cancer cell lines can be injected into syngeneic immunocompetent mice. In most cases, these are subcutaneously injected into the flank of the mouse, allowing easy tumor monitoring by palpation or visual inspection. However, the resulting models do not mimic the growth of tumor cells in their organ of origin. Therefore, orthotopic transplantations became the gold standard for allograft models.
Mouse orthotopic allografts have several advantages: they are cost-effective, can be generated with a relatively simple procedure, and result in models with known molecular makeup, as well as a reproducible and predictable tumor progression and phenotype. Indeed, while patient-derived xenograft models represent the behavior of human PDAC cells accurately, the need for implantation into immunodeficient mice to avoid graft rejection limits the analysis of the tumor-immune and tumor-stroma interactions, allowing researchers to capture only a partial image of the complexity of these tumors. Syngeneic orthotopic allografts of PDAC hold an advantage in this regard also in comparison to genetically engineered mouse models (GEMMs). GEMMs accurately recapitulate human PDAC tumorigenesis and the heterogeneity observed in PDAC patients. However, because of these features, GEMM tumors can show high variance in their genetic makeup, tumor progression, aggressiveness, histological differentiation, and TME composition. While this can be an advantage in certain studies, it limits genotype-to-phenotype studies and the focused investigation of PDAC phenotypes13. Therefore, mouse orthotopic allografts constitute a good tradeoff and model to perform tumor-host and treatment studies in vivo. This paper outlines a protocol for orthotopic transplantation experiments of murine PDAC cells into the mouse pancreas.

<table><tbody><tr><td>27 G cannula</td><td>B.Braun</td><td>08915992</td><td></td></tr><tr><td>Atipamezole (Antisedan 5 mg/mL)</td><td>Orion Corporation</td><td>23554.00.00</td><td></td></tr><tr><td>Autoclip Stainless Steel Wound Clips, 9 mm</td><td>Braintree Scientific</td><td>NC9334081</td><td></td></tr><tr><td>Dulbecco`s Modified Eagle Medium&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D5796-500ML</td><td></td></tr><tr><td>Eye cream (Bepanthen)</td><td>Bayer Vital GmbH</td><td>1578675</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>S0615</td><td></td></tr><tr><td>Fentanyl (50 &amp;micro;g/mL)</td><td>Eurovet Animal Health BV</td><td>9113473</td><td></td></tr><tr><td>Flumazenile (Flumazenil-hameln 0.1 mg/mL)</td><td>Hameln pharma</td><td>09611975</td><td></td></tr><tr><td>Medetomidine (Sedator 1 mg/mL)</td><td>Eurovet Animal Health BV</td><td>400926.00.00</td><td></td></tr><tr><td>Meloxicam (Metacam 5 mg/mL)</td><td>Boehringer Ingelheim Vetmedica GmbH</td><td>3937902</td><td></td></tr><tr><td>Microliter syringe</td><td>Hamilton</td><td>HT80908</td><td></td></tr><tr><td>Midazolam (5 mg/mL)</td><td>Hexal</td><td>00886423</td><td></td></tr><tr><td>NaCl</td><td>B. Braun</td><td>2737756</td><td></td></tr><tr><td>Naloxone (Naloxon-hameln 0.4 mg/mL)</td><td>hameln pharma</td><td>04464535</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P7059-1L</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333-100ML</td><td></td></tr><tr><td>Suture (Ethilon)</td><td>Ethicon</td><td>9999034</td><td></td></tr><tr><td>TrypZean Solution 1x</td><td>Sigma-Aldrich</td><td>T3449</td><td></td></tr></tbody></table>

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.

In the context of a large-scale drug-response study, we successfully implanted more than 170 mice (C75Bl6/J recipient mice, male and female mice sex matched to the PDAC cell lines injected) using the above-described protocol, exemplified in its main steps in Figure 112. In this protocol, we orthotopically implanted three KrasG12D-driven PDAC cell lines (PDAC 1 and 2: Ptf1aCre/+;LSL-KrasG12D/+;LSL-Trp53R172H/+</e...

Watch this Scientific Journal Video about Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer at JoVE.com

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

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, ...

syngeneic-mouse-orthotopic-allografts-to-model-pancreatic-cancer

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

2.6K Views.  German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK). 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. 

Video: Syngeneic Mouse Orthotopic Allografts to Model 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.

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 ...

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 ...

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 ...

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

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

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 ...

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.bdigitaluss.remotexs.co/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 ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Summary

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Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

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

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

A Preclinical Murine Model of Hepatic Metastases

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

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