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 components⁴. Differences in the composition of the TME influence disease prognosis and response to therapy^4,5,6. 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 therapies^{7,8,9,10,11,12}. 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.

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 and genetic background of the animals from which the cell line used for implantation originated (syngeneic allografts).
3. Prepare the mice for implantation.
   NOTE: Handle the mice according to the hygienic standards of the local animal facility.
   1. On the day of implantation, do not feed the mice 4 h before the procedure to avoid complications due to the administration of anesthetics.
   2. Weigh the mice prior to implantation to calculate the amount of the necessary anesthetics.
   3. Transfer the mice to the operation room.
4. Select the PDAC cell lines.
   1. 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: Ptf1a^Cre/+;LSL-Kras^G12D/+;LSL-Trp53^R172H/+, PDAC 3: Ptf1a^Cre/+;LSL-Kras^G12D/+;LSL-Trp53^R172H/R172H).
   2. Select the cell lines that do not express proteins that are potentially immunogenic.
5. 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.
   1. 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's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) and 0.1% penicillin-streptomycin (PS). Transfer the cell suspension to a 25 cm² flask.
   2. Passage the cell lines at least once before implantation.
      1. 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 °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 cm² flask.
         NOTE: As FBS inactivates trypsin, it would hinder trypsinization.
   3. Ensure confluency of ~70%-80% on the day of implantation.
6. Material needed for implantation
   1. Autoclave all the surgical instruments.
   2. Keep anesthetics ready based on the animal experiment license.
      1. Use meloxicam for analgesia at least 5 min in advance as a long-lasting analgetic with anti-inflammatory properties.
      2. For surgery, use a combination of midazolam, medetomidine, and fentanyl (MMF) for analgosedation.
      3. Use a combination of atipamezole, flumazenil, and naloxone (AFN) for antagonizing the MMF analgosedation after implantation.
   3. 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 µL injection volume syringe, and tape.

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.

1. Warm all the liquids to 37 °C before use.
2. 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.
3. Spin down the cells at 200 × 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 µL of the cell suspension to count the cells using a Neubauer chamber and calculate the number of available cells.
4. Dilute the cells to the final required number of cells for injection (e.g., 2,500 cells in 20 µL) 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.

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.

1. Use meloxicam as a perioperative analgesic and apply 5 mg/kg body weight subcutaneously.
2. 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]).
3. Put the mouse back into the cage for 10-15 min.
4. Apply eye cream when the mouse is asleep.
   1. 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.
5. 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.
6. 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.
7. Using scissors and forceps, separate the skin from the peritoneum to get an easier access to the peritoneum.
8. Change the pair of scissors. Open the peritoneum at the location of the spleen with a 1 cm longitudinal cut.
9. 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.
10. Carefully pull the pancreas out of the incision to enable easier access to the organ'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's capsule is held under tension at the site of needle penetration to avoid spillage.
11. Use the dominant hand to carry out the injection. Carefully penetrate the organ's capsule with the syringe'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 µL of DMEM) with a 27 G cannula and a 50 µL syringe into the tail area of the pancreas.
    NOTE: A transparent bubble forms upon successful injection into the tissue.
12. 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.
13. 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.
14. 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.
15. If necessary, prevent dehydration of the animals due to the procedure and the anesthesia by injecting 0.5 mL of sodium chloride subcutaneously.
16. 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.
17. Place the mouse in a 37 °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.

4. Aftercare for the mice

1. Give the mice access to water and food when back in the animal facility.
2. Continue subcutaneous or oral application of meloxicam (5 mg/kg) every 6-12 h for 2-3 days after surgery.
3. Monitor the health status of the mice regularly according to the animal protocol and the institutional guidelines.
4. 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).
   ​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.

5. Harvesting tumor grafts

1. 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.
2. 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.
3. Place the mouse supine on a sterile mat and tape its legs to the surface. Use 80% ethanol to disinfect the abdomen.
4. Wear new gloves and disinfect them. Use surgical scissors to cut the skin longitudinally on the central abdomen.
5. Using scissors and forceps, separate the skin from the peritoneum to get an easier access to the peritoneum.
6. Change the pair of scissors. Open the peritoneum at the central abdomen with a 5 cm longitudinal cut.
7. 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.
8. Store the tumor tissue in a medium suitable for downstream experiments. Process the tissues immediately or, if necessary, keep the sample at 4 °C for a maximum of 6 h until further processing.

Representative 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/+, PDAC 3: Ptf1a^Cre/+;LSL-Kras^G12D/+;LSL-Trp53^R172H/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.

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 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 elsewhere^13,15. 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.

Materials

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