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

  • 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 1: 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, )
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 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 ( protocol 1, steps 1 to 21)

Basic Protocol 3: Use of the Orthotopic PDAC Model in Cancer Cachexia Studies

  • Mice bearing PDAC tumors (from step 23 of 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, ), implanting pancreatic tumor cells in mice ( protocol 1), and evaluating PDAC model (Basic Protocols protocol 22 and protocol 33)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 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

Videos

Literature Cited

Literature Cited
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