Pancreatic Acinar-to-Ductal Metaplasia and Pancreatic Cancer

Liang Wang, Dacheng Xie, and Daoyan Wei


Acinar-to-ductal metaplasia (ADM) of the pancreas is a process that pancreatic acinar cells differentiate into ductal-like cells with ductal cell traits. The metaplasia of pancreatic acinar cells manifests their ability to adapt to the genetic and environmental pressure they encounter. However, with oncogenic genetic insults and/or sustained environmental stress, ADM may lead to pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic cancer. Understanding the intermediate states of ADM and important molecules that regulate ADM formation may help the development of novel preventive strategies that could be translated to the clinic to benefit the people with high risk of pancreatic cancer. Mouse model is widely used in both in vivo and ex vivo studies of ADM. In this chapter, we describe detailed protocols of injury models of the adult mouse pancreas that can function as a tool to study mechanisms of ADM formation.

Key words Acinar-to-ductal metaplasia, Pancreatic ductal ligation, Pancreatitis, Explant culture, Pancreatic cancer

1 Introduction

Pancreatic ductal adenocarcinoma (PDA) remains one of the most deadly human malignancies [1]. Understanding the molecular mechanisms behind pancreatic cancer initiation has significant impact on early detection and effective prevention of this disease to improve the outcome of PDA. Over the past few decades, tremendous efforts have been made to define the pancreatic cell types responsible for tumor initiation [2, 3]. Pancreatic intraepithe- lial neoplasia (PanIN), considered as the main pathological basis of PDA development, display properties of ductal cells as well as tumor cells in PDA [4]. It was initially assumed that PDA originates from any of the ductal cell types that populate pancreatic tissue. However, with the establishment of genetically engineered mouse models (GEMM) of pancreatic cancer,accumulating evidence now- adays supports acinar cells as the main cellular origin of PDA, while acinar-to-ductal metaplasia (ADM or acinar to ductal reprogram- ming) is generally believed to be the precursor lesion of PanIN (Fig. 1) [5, 6]. ADM is the very early histologic lesion observed in PDA animal models and human pancreatic tissue samples.

Fig. 1 H & E staining of pancreatic tissue section derived from Pdx-Cre; LSL-KrasG12D mouse shows intermediate states of acinar-to-ductal metaplasia. A normal acinar structure is marked by yellow arrow. An ADM structure containing both acinar (zymogen granules, intense eosinophilic staining, black arrows) and duct-like cells with a mucinous cytoplasm (black arrow heads) is shown in the middle. Typical PanIN lesions are marked at the bottom

The metaplasia is generally considered as a transdifferentiation of one differentiated cell type to another differentiated cell type. Consistently, acinar cells following ADM formation show increased expression of ductal cell markers, such as cytokeratin-19 (CK-19) or Sex-determining region Y box 9 (SOX9) and decreased expres- sion of acinar cell markers, such as amylase or Mist-1 [7, 8]. The metaplasia of pancreatic acinar cells displays their plasticity nature, which may represent a host intrinsic defense mechanism to protect acinar cells from damage under genetic and/or environmental pressure given that acinar cells are much more sensitive to adverse stimuli than other lineage cells in the pancreas. ADM may be reversible if the cellular pressure is quickly resolved. However, acinar cells under sustained stress, particularly in the presence of mutant KRAS or persistent aberrant growth factor signaling plus stress and/or injury, may lead to irreversible cellular identity change (transdifferentiation) and progression to PanIN. The process of ADM is regulated by both transcriptional and epigenetic mechan- isms. Some transcription factors controlling pancreatic duct devel- opment, such as SOX9 and hepatocyte nuclear factor 6 (HNF6), or transcription factors critical for somatic stem cell reprogramming, like KLF4, have been demonstrated to regulate ADM formation under pathological conditions [5, 6, 9].

Currently, there are many fundamental questions remain unan- swered regarding ADM and pancreatic cancer initiation, such as how acinar cells sense the genetic and environmental stress? What are the determinant factors that drive acinar-to-ductal cell repro- gramming? How do acinar and/or ductal cells maintain their cellu- lar identity? To address those important questions, researchers need appropriate in vivo and ex vivo ADM models. This chapter presents detailed experimental methods to induce ADM by pancreatic duc- tal ligation (PDL), caerulein induced pancreatitis mouse model, and acinar cell explant culture. PDL is the surgery of partial ligation of mouse main pancreatic duct that results in an obstruction of drainage of pancreatic juice out of the tail region of the pancreas. The inflicted damage induces acinar atrophy and ADM. Caerulein treatment is a method to induce acute pancreatitis that facilitates ADM formation. Explant culture is cell culture based method to induce and study AMD in ex vivo.

2 Materials
2.1 Induction of ADM in Mouse Model
2.1.1 Pancreatic Ductal Ligation

1. Autoclaved surgical tools, including scalpel, scissors, tweezers, etc.
2. Buprenorphine.
3. Ketamine hydrochloride and Xylazine (Sigma-Aldrich).
4. Chlorhexidine digluconate solution (Sigma-Aldrich) and 70% alcohol solution.
5. Duratears Eye Ointment (Alcon).
6. Hot bead sterilizer.
7. Electronic shaver or safety razor.
8. Sterile phosphate-buffered saline (PBS) solution.
9. 6-0 and 4-0 prolene suture (Ethicon)
10. Heating pad and paper bedding.

2.1.2 Caerulein Treatment

1. Caerulein (Sigma).
2. Syringe and 27G needle.

2.2 Explant Culture of Mouse Pancreatic Epithelial Cells

1. Collagenase-P (Boehringer Mannheim, Mannheim, Germany).
2. Hanks balanced salt solution.
3. 100-μm Steriflip Nylon filter (Millipore)
4. RPMI-1640 Medium and HEPES Buffer Solution (Sigma- Aldrich).
5. Soybean trypsin inhibitor (SBT1) (Sigma-Aldrich).
6. Dexamethasone (Sigma).
7. Penicillin G and streptomycin antibiotics (Life Technologies, Carlsbad, CA).
8. Rat tail collagen type I (RTC) (Collaborative Biomedical Pro- ducts, Bedford, MA).
9. 24-well culture plate (Corning, Corning, NY)
10. Recombinant TGF-α (R&D Systems, Minneapolis, MN).
11. 4% paraformaldehyde solution (Affymetrix/USB)
12. Anti-CK19 antibody (TROMA-III, DSHB).

3 Method
3.1 Induction of ADM in Mouse Models
3.1.1 Induction of ADM by Pancreatic Ductal Ligation

1. Prepare all supplies and tools for the surgery using proper aseptic technique. Provide a heating pad at a temperature of 38 ◦C to keep mouse body temperature during surgery. Pre- pare a recovery area consisting of a large cage, lined by flat paper bedding (see Note 1).
2. 8- to 10-week-old mice are used for pancreatic ductal ligation. Buprenorphine is used as analgesia (0.05–0.1 mg/kg) 30 min prior to surgery.
3. Anesthetize the mice by intraperitoneal injection of 100 mg/ kg of ketamine and 5–16 mg/kg of xylazine (see Note 2). An efficient anesthetization is indicated by gradual loss of volun- tary movement and muscle relaxation. Test the loss of reflexes by toe pinching (see Note 3).
4. Disinfect thorax and abdomen with antiseptic chlorhexidine solution. Use an electronic shaver or a manual safety razor to shave an area of 2.5 cm × 1.5 cm of the abdomen. Then disinfect the shaven area using gauze soaked with 70% alcohol solution.
5. Position the mouse in the surgical area so that the prepared surgical site is upwards facing the surgeon. Drape the mouse using a waterproof surgical drape with an open window that exposes the disinfected abdominal region. Make sure the rest of the mouse body is covered to create a sterile working field.
6. A laparotomy is made using a sterile scalpel through a midline abdominal incision. Separate the underlying linea alba and the peritoneum using sterile scissors to expose the upper abdomi- nal cavity.
7. Using sterile tweezers to retract the stomach superiorly, expos- ing the spleen and the splenic lobe (the tail region) of the pancreas (see Note 4).
8. To expose the head, neck, and body region of the pancreas for ligation, gently retract the duodenum and part of the upper jejunum to the right upper abdominal cavity.
9. Use 6–0 prolene thread to ligate the pancreatic main duct in the neck region so that the gastric (head) and the splenic (tail) parts of the pancreas are separated (see Note 5).
10. Perform a second ligation around the pancreas, just next to the blood vessels, in a region close to the great curvature of the stomach marked by the cranial mesenteric lymph nodes.
11. Place the organs back into the abdominal cavity. Close the incision using 4–0 prolene thread in a continuous suture pat- tern for the muscle/peritoneal layer and in a discontinuous suture pattern for the skin.
12. When the surgery is complete, place the mouse in the recovery cage with a heating pad and flat paper bedding in order to maintain normal body temperature. Do not return animals to the animal holding area until all animals appear normal. Any animal that has had surgery must have regained the ability to right itself in the cage and be able to move about normally before being returned to the holding area.
13. Use buprenophine as analgesia (0.05–0.1 mg/kg) twice daily for 2 days post-surgery. During the entire experiment, monitor animals periodically for food/water intake recovery and unex- pected signs of illness or infection.
14. At postsurgical day 5 to 7, euthanize mice and open abdominal cavity to obtain good access to the pancreas. The ligated tail portion of the pancreas now has reduced size and become almost translucent with islets that are visible as small white dots. The unaffected head portion of the pancreas is opaque pink and distinct exocrine glands can be observed.
15. To collect protein and/or RNA samples, cut pancreatic tissues from tail and head regions respectively, excluding the ligature and the tissue immediately adjacent to it to avoid cross con- tamination. To evaluate the success of pancreatic ductal liga- tion, cut the ligature portion that contains both unaffected and ligated pancreatic tissues on each side. After histological pro- cessing and H&E staining, the ligated portion of pancreas has reduced number of acinar cells, and duct-like structures (ADM) are observed under microscope (Fig. 2).

3.1.2 Induction of ADM by Caerulein Treatment

1. 6- to 8-week-old mice are divided into phosphate-buffered saline (PBS)-injected control group and caerulein treated experimental group. If necessary, the experimental group can include more mice to perform dose responsive and/or time course responsive studies.
2. Caerulein is dissolved in PBS solution and the working con- centration is 10 μg/mL. Intraperitoneally inject caerulein to mice hourly for consecutive 9 h each day and for two consecu- tive days at the dose of 50 μg/kg body weight per injection.

Fig. 2 On day 5 after pancreatic ductal ligation, mouse pancreatic tissue section is stained by H & E. Many duct-like structures (blue arrow heads) are observed in the ligated portion (right of the dash line), while a few residual acini are also observed (yellow arrows). A lot of normal acini (red arrowheads) are observed in the unaffected portion (left of the dash line)

The control mice are injected with PBS following the same procedure.
3. Inspect the mice hourly for signs of ataxia or other signs of disturbance within the period of injection and three times daily thereafter.
4. The day of the final injection is defined as day 0. At day 3, the first batch of control and experimental mice are euthanized and the pancreatic tissues from the mice are collected for different histological staining. ADM-like lesions of pancreatic acini after caerulein-induced acute pancreatitis can be easily observed in the experimental group while the pancreas tissues of control mice are generally normal (Fig. 3).
5. At day 20, the pancreas samples from the second batch of control and experimental mice are collected and stained as previously described. At this time, an almost complete mor- phologic recovery of the pancreas tissues from experimental mice is observed that no significant differences can be distin- guished between the control and experimental groups.

3.2 Induction of ADM from Explant Culture of Mouse Pancreatic Cells

1. Anesthetize 6-week-old mice as it is described in Subheading 3.1.1, and then open abdominal cavity and cut off the perito- neovenous vein for bleeding using a sterile scalpel and scissors.
2. Use 2 to 3 mL of collagenase-P digestion solution (0.2 mg/ mL) to perfuse from the heart, and quickly remove the whole pancreas and briefly wash it for several times in Hanks balanced salt solution.
3. In a sterile biosafety cabinet, transfer the pancreas into a
60 × 15 mm cell culture dish containing 1 mL of collagenase-P digestion solution and mechanically mince pan- creas into small pieces using a sterile scalpel and forceps (see Note 6). Incubate the culture dish at 37 ◦C with 5% CO2 in air for 15 min with occasional shaking every 3 min.
4. Sequentially filter collagenase-digested pancreatic tissues through a 100-μm nylon filter (Fig. 4). The filtrate is passed through a 30% fetal bovine serum cushion at 1000 rotations per minute.
5. The cellular pellet is washed twice with and suspended in conditional RPMI1640 medium containing 15 mM HEPES,
0.1 mg/mL SBT1 (soybean trypsin inhibitor), 1 μg/mL dexa- methasone, 1% fetal bovine serum (see Note 7), penicillin G (1000 U/mL), and streptomycin (100 μg/mL) antibiotics.
6. Add an equal volume of neutralized rat tail collagen type I (RTC) to the cellular suspension. Pipette the cellular/RTC
mix suspension (500 μL) into each well of a 24-well culture plate precoated with 200 μL of RTC.
7. After solidification of the RTC, add additional conditional RPMI1640 media. Cultures are maintained at 37 ◦C and 5% CO2 in air for up to 7 days.
8. Explants harvested from mice pancreas are maintained in the absence (control group) or presence of recombinant TGF-α (25 ng/mL) to induce ADM formation. Media supplemented with appropriate growth factors and inhibitors are exchanged on day 1 and day 3.
9. At day 3–7, the explant cultures will develop a ductal epithelial morphology characterized by large cystic structures lined by cuboidal and simple squamous epithelia (Fig. 5). The ductal- like morphology coincides with overexpression of CK-19, a ductal epithelial-specific marker. For detection of CK-19 expression, whole collagen gels are fixed in 4% paraformalde- hyde, followed by immunocytochemistry staining with anti- CK19 antibody.

Fig. 3 On day 3 after Caerulein treatment, pancreatic tissue sections from control (PBS injection) and experimental mice are stained by H & E. ADM-like structures with dilated lumens (green arrows) are observed in caerulein treated mouse pancreas (right panel), while normal acini are observed in control mouse pancreas (left panel)

Fig. 4 Digested pancreatic tissues are passed through a 100-μm polypropylene filter (left panel), and filtered cells are suspended in conditional RPMI1640 medium (right panel) for in vitro explant culture

4 Notes

1. All studies related to animal manipulation and surgeries should follow the applicable laws and regulations of the institution and/or country in which the research is conducted.
2. Inhaled anesthetic agents can also be used for mouse surgery. For example, isoflurane/sevoflurane can be used as an anes- thetic agent when it is administered with a properly calibrated vaporizer.
3. Apply ophthalmic ointment to prevent dryness of the eyes when mice are under anesthesia.
4. To prevent drying-out of the exposed internal organs, regularly sprinkle them with sterile PBS solution.
5. Conduct the ligation very carefully not to damage the underly- ing blood vessels, such as the superior pancreaticoduodenal artery, the inferior pancreaticoduodenal artery and the splenic artery. In addition, do not use too much force to avoid com- plete ligation of the pancreatic main duct, which may cause acute necrosis of the ligated tail part of the pancreas and death of mouse.
6. Additional trituration step and digestion solution may be needed until the tissue is completely dissociated so that they are small enough and do not clog the 5-mL pipet.
7. To minimize the effects of other growth factors on ADM induction, the explants are cultured in a low (1%) and heat- inactivated serum environment.

Fig. 5 Representative images of explant culture of mouse pancreatic acinar cells for days 3 and 5, respectively. Ductal-like cell clusters are formed when treated with TGF-α. The sizes of ADM-like clusters grow from day 3 to day 5


We thank Professor Keping Xie for support. This work was sup- ported in part by grants R01-CA129956, R01-CA148954, R01-CA152309, R01-CA172233, R01-CA195651,
R01-CA198090, and R01CA220236 from the National Cancer Institute, National Institutes of Health, and from the
M.D. Anderson Cancer Center Institutional Research program. All authors disclose no conflicts of interest.


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