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 ...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimated+projection+of+US+cancer+incidence+and+death+to+2040.">Estimated projection of US cancer incidence and death to 2040.</a> JAMA Network Open. 4 (4), 214708 (2021).</li><li>Schneider, G., Schmidt-Supprian, M., Rad, R., Saur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+tumorigenesis:+Context+matters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Hedgehog+signaling+enhances+delivery+of+chemotherapy+in+a+mouse+model+of+pancreatic+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-induced+vascular+remodeling+creates+therapeutic+vulnerabilities+in+pancreas+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics-driven+precision+medicine+for+advanced+pancreatic+cancer:+Early+results+from+the+COMPASS+trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+phenotypes+of+pancreatic+cancer+are+driven+by+genomic+events+during+tumor+evolution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+classification+of+pancreatic+ductal+adenocarcinoma+that+predicts+molecular+subtypes+and+correlates+with+clinical+outcome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unifying+paradigm+for+transcriptional+heterogeneity+and+squamous+features+in+pancreatic+ductal+adenocarcinoma.">A unifying paradigm for transcriptional heterogeneity and squamous features in pancreatic ductal adenocarcinoma.</a> Nature Cancer. 1 (1), 59-74 (2020).</li><li>Mueller, S., 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=Evolutionary+routes+and+KRAS+dosage+define+pancreatic+cancer+phenotypes.">Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.</a> Nature. 554 (7690), 62-68 (2018).</li><li>Falcomata, C., 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=Selective+multi-kinase+inhibition+sensitizes+mesenchymal+pancreatic+cancer+to+immune+checkpoint+blockade+by+remodeling+the+tumor+microenvironment.">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><li>Erstad, D. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 &#181;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, ...

This protocol provides reproducible, immunocompetent PDAC in vivo models suitable for genotype, phenotype, and drug response studies. The main advantage of this technique is its ability to help investigate tumor progression with a syngeneic biological tumor microenvironment while maintaining the reproducibility of the experiment. Demonstrating the procedure will be Stefanie Barthel and Chiara Falcomata, both are from our laboratory.Begin the preparation of the tumor cells by washing the pancreatic ductal adenocarcinoma, or PDAC cells, grown in a T75 culture flask with phosphate buffered saline or PBS. Then add 0.5 milliliters of trypsin for five to 10 minutes to detach from the flask. Re-suspend the detached cells in 10 milliliters of DMEM with 10%fetal bovine serum, or FBS, and 0.1%penicillin streptomycin before transferring the cell suspension to a 15 milliliter tube.Centrifuge the cell suspension at 200G for five minutes and aspirate the supernatant. Re-suspend the cell pellet in DMEM without additives, based on the required final concentration of the cells for injection. Count the cells in 10 microliters of cell suspension, using the Neubauer Chamber and calculate the number of available cells.Transfer one milliliter of the diluted suspension, as per the final required concentration, to a 1.5 milliliter tube. Place the tube on a rotator until implantation to prevent cell aggregation. For orthotopic implantation of PDAC cells, apply eye cream to the analgosedated mouse while the mouse is sleeping.Check the sufficient analgosedation before shaving the left lateral abdominal flank of the analgosedated mouse. Place the mouse, lying on the right side, on a sterile heating mat and tape the legs to the surface. Wear new gloves and disinfect them.Using surgical scissors, cut about one centimeter of the skin and subcutaneous fat tissue longitudinally on the left flank where the spleen is projected. Using scissors and forceps, separate the skin from the peritoneum to get easier access to the peritoneum. Change the pair of scissors before opening the peritoneum at the location of the spleen with a one centimeter longitudinal cut.While mobilizing the spleen and the attached pancreas using blunt, fine tipped forceps, ensure that the organs are not attached to other tissue. Also check for the supplying blood vessels to avoid injuring the vessels or the surrounding tissue when handling the pancreas. Carefully pull the pancreas out of the incision to enable easier access to the organ's tail and observe the spleen following the pancreas.Ensure that the organ is not folded during injection and the organ's capsule is held under tension at the needle penetration site to avoid spillage. Aim for a piece of pancreatic tissue between the visible blood vessels to minimize the risk of puncturing or injecting the vessel. Using the dominant hand, carefully penetrate the organ's capsule with a 27 gauge cannula and 50 microliter syringe following the longitudinal axis of the organ.After injecting the 20 microliters of suspension containing 2, 500 cells into the tail area of the pancreas, keep the pancreas and inserted syringe still for approximately one minute to avoid spilling the tumor cells. Then, remove the needle from the injection site. Next, carefully rearrange the organs inside the abdomen without injuring the tissue to avoid spilling the cells.Avoid organ adhesion by pouring one milliliter of 0.9%sodium chloride onto the organs. After suturing the peritoneum using the simple interrupted suture technique, close the skin using two to three nine millimeter stainless steel wound clips. Then antagonize the analgosedation with midazolam, medetomadine, and fentanyl, or MMF, with a subcutaneous injection of AFN according to the body weight of the mouse.Once done, place the mouse in a 37 degree Celsius heated chamber until it is awake and completely active. Place the active mouse back into its original cage and monitor the health status of the mouse by checking the fur, skin color, and activity, and repeat it three to four hours after surgery. In MRI of implanted mouse, the engraftment of PDAC cells was abdominally palpated earliest at approximately seven days post-surgery, depending on the cell number injected and the aggressiveness and growth characteristics of the inoculated cell line.The survival of the resulting implanted mice varied depending on the tumor cell intrinsic features of the cell lines. Successful intra-pancreatic injections led to full-blown tumors in the mouse pancreas. In contrast, unsuccessful implantations resulted in the absence of pancreatic tumors because of injection of non-viable cells, rejection of the implanted cells, or injection of an insufficient number of cells.Remember to pay attention to not spill any cells during injections and not to injure any surrounding tissue to prevent cells from spreading inside the abdominal. Following this procedure, in vivo studies, like preclinical drug response studies, can be performed in immunocompetent mice, followed by, for example, facts, sequencing, or histological analysis to investigate tumor host interactions.

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Research

JoVE Journal

Cancer Research

2.3K Aufrufe.  German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK). Dieses Protokoll bietet reproduzierbare, immunkompetente PDAC-In-vivo-Modelle, die für Genotyp-, Phänotyp- und Arzneimittelreaktionsstudien geeignet sind. Der Hauptvorteil dieser Technik besteht darin, dass sie die Tumorprogression mit einer syngenen biologischen Tumormikroumgebung untersuchen kann, während die Reproduzierbarkeit des Experiments erhalten bleibt. Stefanie Barthel und Chiara Falcomata, beide aus unserem Labor, führen das Verfahren vor.Beginnen Sie mit der Präparatio...