Name: syngeneic mouse orthotopic allografts to model pancreatic cancer
Uploaded: 2022-10-04T15:00:00
Description: Watch this Scientific Journal Video about Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer at JoVE.com

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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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 (Video) | JoVE 

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

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

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

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