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, 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.
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.
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
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.
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.
4. Aftercare for the mice
5. Harvesting tumor grafts
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/+, PDAC 3: Ptf1aCre/+;LSL-KrasG12D/+;LSL-Trp53R172H/R172H), which were previously generated in the laboratory. The engraftment of PDAC cells can be abdominally palpated earliest at approximately 7 days post surgery, depending on the cell number injected and the aggressiveness and growth characteristics of the inoculated cell line. MRI (Figure 2A), ultrasound, and BLI of the mouse abdomen can be used for quantitative measurements of the tumor volume. Depending on the tumor cell-intrinsic features of the cell lines, the survival of the resulting implanted mice can vary (Figure 2B). Successful intrapancreatic injections lead to full-blown tumors in the mouse pancreas (Figure 2C,D). In contrast, unsuccessful implantations can result in the absence of pancreatic tumors because of a) injection of non-viable cells, b) rejection of the implanted cells (e.g., if the cells were not syngeneic to the recipient mouse), or c) an insufficient number of cells injected. Alternative negative outcomes can lead to the absence of a primary tumor in the pancreas but the presence of a tumor at the peritoneum, corresponding to the site of injection (e.g., through spillage of the tumor cell suspension from the pancreatic injection site).
Figure 1: Schematic representation of the main steps of the protocol. (A) PDAC cells should be passaged at least once prior to implantation. (B) Cells are trypsinized to detach them from the flask, transferred to a 15 mL tube, and counted. (C) The appropriate cell dilution is placed in a tube and brought to the implantation room. (D) The recipient mouse is anesthetized, shaved, and disinfected in the region where the surgery will take place. (E) A 1 cm cut is made to the mouse abdomen corresponding to the pancreas tail location. (F) The desired number of cells is injected into the tail of the pancreas. Abbreviation: PDAC = pancreatic ductal adenocarcinoma. Please click here to view a larger version of this figure.
Figure 2: Examples of successful outcomes upon syngeneic mouse orthotopic allografts. (A) Representative MRI of a mouse 2 weeks after orthotopic transplantation of primary mouse PDAC cells. Dashed outline denotes the tumor. Scale bar = 5 mm. (B) Kaplan-Meier survival curve of three different mouse PDAC cell lines (PDAC 1-3) orthotopically transplanted into the pancreas of syngeneic immunocompetent mice (C57Bl/6J background). (C) Representative image of a tumor isolated at endpoint from the orthotopically injected PDAC1 cell line. Both a pancreatic tumor resulting from a successful intrapancreatic injection of tumor cells and the spleen are shown in the photograph. Scale bar = 5 mm. (D) Barplot depicting the average tumor weights of orthotopically transplanted tumors from PDAC 1-3. Dots represent individual mice. Abbreviations: PDAC = pancreatic ductal adenocarcinoma; MRI = magnetic resonance imaging. Please click here to view a larger version of this figure.
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 simple and fast procedure for the orthotopic implantation of mouse PDAC cell cultures into the pancreas. To obtain reproducible results, several technical considerations must be kept in mind, including the quality of the surgical technique. As surgical practices vary, it is advisable that one person carries out all the orthotopic injections within the same experiment.
The injection of cells into the pancreas should be carried out with caution. Spilled cells, piercing the pancreas, or injuring the tissue can cause the cells to engraft at other organ sites outside the pancreas. This might complicate the clinical evaluation (e.g., size and local infiltration) of the primary tumor, as well as its local and distant metastatic spread. Therefore, it is recommended to document and assess bubble formation and any information about cell spillage for each implanted mouse. Cuts in the skin and peritoneum should be made at an appropriate size to enable optimal access to the pancreas while keeping the wounds as small as possible.
The number of implanted cells can be adjusted depending on the experimental plan. While larger numbers of implanted cells lead to physiologically rapid-growing tumors and fast progression of clinical symptoms, smaller cell numbers increase the risk that tumors will not engraft. Furthermore, small volumes of cell suspensions are recommended as larger volumes (>20 µL) are associated with an increased potential of the formation of a cystic core13. We found that implanting 2,500-5,000 cells in a volume of 20 µL of cell culture medium is an optimal amount for preclinical therapeutic studies by ensuring tumor growth over multiple weeks. However, for other applications, up to 1.6 × 106 cells can be implanted.
In addition to cell culture medium, Matrigel can be used as a nutrient-rich medium to resuspend the PDAC cells, which also increases the viscosity of the injection solution, thereby preventing leakage of the tumor cells13. Injections into the tail area of the pancreas are preferred with this protocol as the pancreas tail is easily accessible and access to the pancreas head is limited. Protocols to successfully inject cells into the pancreas head are described elsewhere13,15. To perform the surgical procedure, mice are kept under analgosedation using a drug combination, including midazolam, medetomidin, and fentanyl, as well as meloxicam as a peri- and postoperative analgesic. Alternatively, isoflurane in combination with appropriate analgesics can be used as inhalant anesthesia under constant flow. The selection of the analgosedation method of choice for the animal license depends on the institutional guidelines and the respective local authorities.
Careful choice of the cell lines and recipient mice is imperative. Indeed, it is important to ensure that the genetic background of the recipient mice matches the genetic background of the selected cell lines to avoid immunogenicity and graft rejection of the PDAC cells. Furthermore, immunogenic exogenous proteins, such as fluorescent reporter alleles or Cas9, expressed by implanted tumor cells influence the host's response and can lead to an immunogenic reaction. Therefore, tumor growth and the composition of the TME can be affected and cause bias.
In comparison to GEMMs, syngeneic orthotopic mouse allografts show a reduced amount of desmoplasia and, in some instances, a lower rate of metastatic dissemination, limiting, in part, the similarity to human tumors. Moreover, the need for surgical intervention increases the complexity of the procedure. Unsuccessful implantations resulting in the absence of pancreatic tumors in the desired location due to non-viable cell injection, the rejection of implanted cells, low numbers of injected cells, and injection leakage may prevent the completion of the experimental procedure.
Despite these limitations, syngeneic orthotopic mouse allograft models of PDAC have been shown to effectively address many of the challenges of studying PDAC and its heterogeneity. With their highly reproducible phenotypes and tumor growth patterns, as well as tumor-TME interactions, they represent an invaluable resource that can be obtained quickly following a relatively simple protocol.
The authors have nothing to disclose.
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.).
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 |