Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Summary

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.

Abstract

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.

Introduction

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 components

Protocol

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

1. Ensure approval of the animal protocol and personnel by the local authorities before starting any animal experimentation.
2. Select recipient mice.
   1. Select mice of similar ages for implantation (C57Bl/6J recipient mice with an age range between 2 months and 4 months).
   2. Ensure that the sex and genetic background of the recipient animals match the sex an....

Results

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 Kras^G12D-driven PDAC cell lines (PDAC 1 and 2: Ptf1a^Cre/+;LSL-Kras^G12D/+;LSL-Trp53^R172H/+

Discussion

Syngeneic mouse orthotopic allografts represent a robust model for preclinical studies due to their cost-effectiveness, reproducibility, and relatively simple experimental procedures^13,15. 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.......

Materials

List of materials used in this article

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