Orthotopic Model of Human Pancreatic Ductal Adenocarcinoma and Cancer Cachexia in Nude Mice

Susan Jones‐Bolin1, Bruce Ruggeri1

1 Cephalon, Inc., West Chester, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.3
DOI:  10.1002/0471141755.ph1403s37
Online Posting Date:  June, 2007
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Abstract

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related deaths in the United States, with a 5-year survival rate of only 2% to 10%. This tumor is aggressive, often metastasizing to distant sites (liver, lung, and adjacent intestines) by the time of diagnosis. Treatment options are limited, and the disease carries a grave prognosis for most patients. An orthotopic model of human PDAC in nude mice provides an excellent way to evaluate the pathogenesis of tumor growth and metastasis in order to develop therapies, to better define the underlying biology of tumor growth and metastasis, and to identify new molecular targets. This unit describes an orthotopic model of human PDAC in athymic nude mice that closely mimics the human condition. It is characterized by diffuse peritoneal, lymphatic, and hepatic metastatic spread and manifestations of a cancer cachexic phenotype.

Keywords: Human pancreatic ductal adenocarcinoma; Athymic nude mice; Orthotopic; Metastasis; cachexia

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Orthotopic Model of Human Pancreatic Ductal Adenocarcinoma in Athymic Nude Mice
  • Basic Protocol 2: Use of the Orthotopic PDAC Model in Pharmacological Efficacy Studies
  • Basic Protocol 3: Use of the Orthotopic PDAC Model in Cancer Cachexia Studies
  • Alternate Protocol: Establishing Cell Lines from PDAC Tumor Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Orthotopic Model of Human Pancreatic Ductal Adenocarcinoma in Athymic Nude Mice

 Materials
  • COLO-357 (obtained through a materials transfer agreement with Duke University) or AsPC-1 human pancreatic carcinoma cell lines (ATCC #CRL-1682)
  • Growth medium (see recipe)
  • 1× trypsin/EDTA (Mediatech) or similar cell-dissociation agent
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Nude mice, female, 6- to 8-weeks old (20 to 25 g; athymic nu/nu or comparable strain) (Charles River Labs)
  • Matrigel synthetic basement membrane (Collaborative Research)
  • Ketamine/xylazine mixture (see recipe)
  • Isoflurane
  • Oxygen source
  • Hemacytometer
  • 1-ml syringes and 27-G needles for injecting xenograft cells
  • Petri dishes, sterile
  • Surgical instruments including (two sets of instruments recommended):
    • 4.5-inch iris scissors, straight, sharp ends
    • Disposable scalpels
    • 4.75-in. Adson forceps or delicate dressing forceps, serrated
    • 4.5-in. tissue forceps, 1×2 teeth
    • 5.5-in. Mayo-Hegar or similar needle holder
  • Circulating water heating pad: Gaymar T-pump (http://www.gaymar.com) or equivalent
  • Nose cone and appropriate anesthesia apparatus for administering isoflurane
  • Sterile gauze sponges (2-in. × 2-in.)
  • Sterile surgical alcohol prep pads, medium
  • Sutures:
    • Prolene 6/0, taper needle, for suturing tissue to pancreas; one pack per five to ten mice
    • Vicryl 5/0 or 6/0, or PDS II 5/0, for closing peritoneal layer; one pack per five to ten mice
  • 9-mm wound clips, with applier and remover
  • Ear punch or tattooing device to identify individual mice, or waterproof colored markers (e.g., Sharpie)
  • Additional reagents and equipment for cell culture (unit 12.1) and euthanasia of mice (Donovan and Brown, 2006)

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Use of the Orthotopic PDAC Model in Pharmacological Efficacy Studies

 Materials
  • Mice with surgically implanted xenograft of primary pancreatic tissue (Basic Protocol 1, steps 1 to 21)

Alternate Protocol: Establishing Cell Lines from PDAC Tumor Tissue

 Additional Materials (see Basic Protocol 1)
  • Mice bearing PDAC tumors (from step 23 of Basic Protocol 1)
  • Growth medium (see recipe)
  • 10% (v/v) DMSO/90% (v/v) FBS
  • 70-µm nylon cell strainers
  • 50-ml conical centrifuge tubes
  • Plunger from 1-ml syringe
  • Refrigerated centrifuge
  • 75-cm2 tissue culture flasks
  • 1.5-ml cryotubes (e.g., Nunc)
  • Nalgene Cryo 1°C Freezing Container
  • Syringe with 30-G needle
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, 2006), implanting pancreatic tumor cells in mice (Basic Protocol 1), and evaluating PDAC model (Basic Protocols 2 and 3)

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.
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Figures

  •  FigureFigure 14.3.1 (A) Site of incision for PDAC model. (B) Position of retracted spleen and pancreas relative to incision. (C) Exteriorized pancreas tissue with moistened gauze. Spleen is showing and tumor tissue has been attached to the exteriorized pancreas.
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  •  FigureFigure 14.3.2 Hemotoxylin and eosin histological sections of a liver metastasis revealing (A) very little normal liver present in this section, as the pancreatic metastatic tissue has displaced the normal liver tissue; (B) primary pancreatic ductal carcinoma (arrows mark islands of tumor cells among normal glandular tissue); (C) peripancreatic (invasive) metastasis (arrows mark islands of tumor cells); and (D) mesenteric lymph node metastasis (arrows mark islands of tumor cells) from orthotopically-grafted COLO-357 human pancreatic carcinoma tissue. Immunostaining for the pancreas-selective antigen CA19-9 may be performed (Friess et al., 1997) to confirm the pancreatic origin of metastatic lesions.
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  •  FigureFigure 14.3.3 (A) Body weights of athymic nude mice implanted with human PDAC tumor tissue and body weights of sham-implanted mice. Mice implanted with human PDAC tumor tissue (indicated by asterisk over the bar) exhibit significant body weight loss as compared to sham-implanted mice (p < 0.05) despite comparable food consumption. (B) Right hind-leg gastrocnemius and soleus muscle weights of athymic nude mice implanted with human PDAC tumor tissue or sham-implanted mice. Mice implanted with human PDAC tumor tissue (indicated by asterisk over the bar) exhibit significant gastrconemius and soleus muscle mass loss as compared to sham-implanted mice (p < 0.05). All mice including sham-implanted, vehicle-treated, and NCE-treated mice are euthanized on day 60 of dosing, and their right hind-leg muscles evaluated.
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  •  FigureFigure 14.3.4 The effects of chronic administration of lestaurtinib and GMZ, separately and in combination, on the survival of nude mice orthotopically implanted with human PDAC tissue. Kaplan-Meier survival curves showing effects of treatment regimens on survival of tumor-bearing mice were analyzed by the Kaplan-Meier method; Mann-Whitney Rank Sum test analyses were used to compare mean and median survival times between treatment groups. Single asterisks signify mean survival significant to vehicle- and lestaurtinib monotherapy–treated mice (p < 0.001). Double asterisks signify mean survival significant to vehicle and lestaurtinib monotherapy–treated mice as well as gemcitabine-treated mice (p < 0.001 and p = 0.04, respectively).
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  •  FigureFigure 14.3.5 Metastatic score of mice orthotopically implanted with human PDAC and treated with CEP-7055 or vehicle. A score of I indicates minimal spread beyond the primary mass while a score of IV indicates widespread peritoneal metastasis. See Basic Protocol 2 for further definition of metastatic scoring criteria.
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  •  FigureFigure 14.3.6 Effects of EPA, infliximab, and bortezomib on muscle weights in an orthotopic model of human PDAC in nude mice. The y axis shows right hind-leg gastrocnemius and soleus muscle weights of athymic nude mice implanted with human PDAC tumor tissue or sham-implanted mice. Bortezomib therapy caused significant increases in gastrocnemius (**p = 0.003) and soleus (**p =< 0.001) muscle weights compared to vehicle-treated mice. EPA therapy also increased gastrocnemius (*p = 0.004) and soleus (*p = 0.018) muscle weights compared to vehicle-treated mice. Infliximab therapy had no effect on muscle weights compared to vehicle-treated mice.
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  •  FigureFigure 14.3.7 Effects of EPA, infliximab, and bortezomib on metastatic scores in an orthotopic model of human PDAC in nude mice, where a score of I indicates minimal spread beyond the primary tumor mass while a score of IV indicates extensive spread. Bortezomib-treated mice had a lower incidence (40%, 4 of 10) of metastatic scores of III or IV compared to vehicle-treated mice (90%, or 9 of 10). Neither EPA nor infliximab therapy had an effect on metastatic scores, with 80% (8 of 10) of the mice having scores of III or IV.
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  •  FigureFigure 14.3.8 Effects of EPA, infliximab, and bortezomib on primary tumor mass weights in an orthotopic model of human pancreatic ductal adenocarcinoma (PDAC) in nude mice. Bortezomib therapy had a significant (*p = 0.024) effect on primary tumor weight compared to vehicle-treated mice. Neither EPA nor infliximab therapies provided any effect on primary tumor weights in this model. Sham tumor weights were not included on the graph because the weights of the normal pancreas plus spleen are very low (~0.2 to 0.3 g).
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Literature Cited

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