The TRAMP Mouse as a Model for Prostate Cancer

Arthur A. Hurwitz1, Barbara A. Foster2, James P. Allison3, Norman M. Greenberg4, Eugene D. Kwon5

1 SUNY Upstate Medical University, Syracuse, New York, 2 University of Pittsburgh, Pittsburgh, Pennsylvania, 3 University of California, Berkeley, Berkeley, California, 4 Baylor College of Medicine, Houston, Texas, 5 Loyola University Medical Center, Maywood, Illinois
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 20.5
DOI:  10.1002/0471142735.im2005s45
Online Posting Date:  November, 2001
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Abstract

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mirrors the pathogenesis of human prostate cancer. Male TRAMP mice uniformly and spontaneously develop autochthonous (orthotopic) prostate tumors following the onset of puberty. Prostate cancer occurs consequent to the expression of SV40 T antigen. The versatility of the TRAMP model has been extended by establishment of several TRAMP‐derived prostate tumor cells lines that can be injected into syngeneic male nontransgenic C57BL/6 hosts to induce ectopic prostate tumorigenesis. Subcutaneous tumor induction using the TRAMP‐C cell lines has provided the basis for two additional murine models, the first of which can be used for rapid screening of experimental therapies for the treatment of primary prostate tumors and the second for testing the effectiveness of adjunctive therapies targeting prostate cancer metastases. Detailed descriptions for the harvesting and microdissection of TRAMP prostates and tumors, and the evaluation and scoring of tumors are also provided.

     
 
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Table of Contents

  • Basic Protocol 1: The Breeding and Screening of C57BL/6 (H‐2b) or C57BL/6 × FVB/N (H‐2q) F1 TRAMP Mice
  • Basic Protocol 2: Syngeneic TRAMP‐C1 and ‐C2 Subcutaneous Tumor Model
  • Alternate Protocol 1: TRAMP‐C2 Subcutaneous Tumor Resection‐Metastasis Model
  • Support Protocol 1: Microdissection of the Mouse Prostate
  • Support Protocol 2: Scoring Tumors in the Autochthonous Tramp Mouse
  • Support Protocol 3: Castration of Mice
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Breeding and Screening of C57BL/6 (H‐2b) or C57BL/6 × FVB/N (H‐2q) F1 TRAMP Mice

  Materials
  • Pure C57BL/6 heterozygous TRAMP colony (Greenberg or Jackson Laboratories; see Internet Resources)
  • 6‐ to 8‐week‐old male nontransgenic C57BL/6 or FVB/N mice (Charles River, Jackson Laboratories, or Taconic)
  • 70% ethanol, 4°C
  • recipeTail lysis buffer (see recipe)
  • 10 mg/ml proteinase K in distilled deionized H 2O
  • 1:1 (v/v) phenol:chloroform
  • 100% ethanol, 4°C
  • recipeTE buffer, pH 8.0 ( appendix 2A)
  • recipePCR master mix (see recipe)
  • 1% agarose gel (unit 10.4)
  • Sharp scissors, clean
  • 2‐ml tube, sterile
  • 55°C incubator
  • Platform rocker
  • Tabletop centrifuge
  • Additional reagents and equipment for anesthesia using methoxyflurane (unit 1.4), mouse euthanasia (unit 1.8), determining DNA concentration and purity by absorption spectroscopy ( appendix 3L), and agarose gel electrophoresis (unit 10.4)
NOTE: For recent updates pertaining to the breeding and screening of TRAMP mice, refer the Greenberg lab homepage located at http://128.249.134.198.

Basic Protocol 2: Syngeneic TRAMP‐C1 and ‐C2 Subcutaneous Tumor Model

  Materials
  • TRAMP‐C1 or ‐C2 cell lines (Greenberg Laboratory; see Internet Resources)
  • recipeTRAMP tissue culture medium (see recipe)
  • 0.05% (w/v) trypsin/0.53 mM EDTA solution (Life Technologies)
  • Dulbecco's Modified Eagle Medium (DMEM; appendix 2A)
  • 6‐ to 8‐week‐old male nontransgenic C57BL/6 mice
  • 10‐cm tissue culture dishes or 75‐cm2 flasks
  • 15‐ or 50‐ml conical tube
  • 1‐ml syringe and 19‐ to 21‐G, 1.5‐in. hypodermic needle
  • Electric clippers (Oster) and size 40 blade
  • Vernier calipers
  • Additional reagents and equipment for counting cells on a hemacytometer ( appendix 3B) and anesthesia using methoxyflurane (unit 1.4)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: TRAMP‐C2 Subcutaneous Tumor Resection‐Metastasis Model

  • C57BC/6 male mice with TRAMPC‐2 neck tumors (see protocol 2)
  • Vernier calipers
  • Metzenbaum scissors (Allegiance Healthcare Company)
  • Michelle skin clips (Becton Dickinson)
  • Additional reagents and equipment for euthanasia by CO 2 asphyxiation (unit 1.8), removal of the lymphoid organs (unit 1.9), paraffin embedding and sectioning (unit 21.4), and hematoxylin and eosin staining (unit 12.8)

Support Protocol 1: Microdissection of the Mouse Prostate

  Materials
  • Male TRAMP mouse (see protocol 1)
  • 70% ethanol
  • Dissection medium, 4°C: cell culture media (e.g., DMEM) or balanced salt solution—i.e., phosphate buffered saline ( recipePBS) or Hank's balanced salt solution ( recipeHBSS; appendix 2A)
  • Small scissors
  • Two grade‐4 forceps
  • Small Vannas scissors
  • Serrated curved forceps
  • 10‐cm petri or cell culture dish
  • Stereo dissecting microscope
  • High‐intensity lamp
  • Additional reagents and equipment for anesthesia using methoxyflurane (unit 1.4) and euthanasia by exsanguination (unit 1.8)
NOTE: Lobes are placed into individual tissue cassettes or molds for either fixation or snap freezing, respectively. Alternatively, tissue can be transiently kept in 4°C DMEM until transfer into cassettes or molds.NOTE: For more information concerning the anatomy of the mouse urogenital tract, the reader is referred to the Greenberg Laboratory homepage (http://128.249.134.198; see Internet Resources).

Support Protocol 2: Scoring Tumors in the Autochthonous Tramp Mouse

  Materials
  • Microdissected prostate lobes (see protocol 4)
  • Additional reagents and equipment for paraffin embedding and sectioning (unit 5.8), and hematoxylin and eosin staining (unit 12.8)

Support Protocol 3: Castration of Mice

  Materials
  • 12‐week‐old male TRAMP mice (see protocol 1)
  • 10% povidone‐iodine
  • Iris scissors
  • Electrocautery device (Roboz)
  • 2 small hemostats (optional)
  • Michelle surgical clips (Response) or nonabsorbable suture
  • Additional reagents and equipment for anesthesia using methoxyflurane (unit 1.4)
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Figures

  •   FigureFigure 20.5.1 The reproductive tract in an adult male mouse. The arrow indicates where to cut the urethra when removing the reproductive tract. The asterisk (*) indicates the connective tissue between the body wall and the bladder. Abbreviations: AP, anterior prostate; BL, bladder; SV, seminal vesicles; VD, vas (ductus) deferens; VP, ventral prostate.
  •   FigureFigure 20.5.2 Location of the draining lymph nodes. The reproductive tract has been removed from an adult male mouse revealing the draining lymph nodes (indicated by arrows). They are located on either side of the abdominal aorta above its bifurcation into the iliac arteries. Abbreviations: A, aorta; I, iliac vessels; kid, kidney; VC, vena cava.
  •   FigureFigure 20.5.3 Ventral view of an adult male mouse genitourinary complex. This complex has been removed from an adult male mouse and placed in dissecting media. Abbreviations: AP, anterior prostate; BL, bladder; LP, lateral prostate; SV, seminal vesicles; VD, vas (ductus) deferens; VP, ventral prostate.
  •   FigureFigure 20.5.4 The ventral and lateral lobes of the prostate. Connective tissue and fat surrounding the ventral and lateral prostate have been removed. The ventral and lateral lobes of the prostate have been surgically separated and their points of insertion are now visible. Abbreviations: BL, bladder; LP, lateral prostate; SV, seminal vesicles; VP, ventral prostate.
  •   FigureFigure 20.5.5 The dorsal view of an adult male mouse prostate. The forceps demonstrate the location of the dorsal prostate just below the seminal vesicles on the posterior aspect of the reproductive tract. The ampullary gland is visible between the seminal vesicles. Abbreviations: amp gl, ampullary gland; BL, bladder; DP, dorsal prostate; SV, seminal vesicles.
  •   FigureFigure 20.5.6 Dissection of the ampullary gland. The ampullary gland is located at the base of the ductus deferens and must be removed with scissors. The arrow indicates the cut end of the urethra. Abbreviations: amp gl, ampullary gland; BL, bladder; DP, dorsal prostate; VD, vas deferens.
  •   FigureFigure 20.5.7 Histologic grades of TRAMP tumors. TRAMP tumors were microdissected and prepared for routine histopathologic analysis. The numbers indicate the score corresponding to the histologic grade of the lesion depicted by each arrow. As shown in panel (A), early prostatic lesions can consist of a variety of histologic grades, ranging from normal epithelium (1) to low grade PIN (2), or more advanced PIN (3). The lumen (L) of a single acinar structure is indicated. More advanced lesions are detected in older mice. In panel (B), the initial stages of adenocarcinoma (4) are represented by a narrowing or loss of interductal spaces (IS) and substantial reduction in the size of acinar lumens. In panel (C), more advanced adenocarcinoma (5) is characterized by loss of lumenal and ductal structures and a less‐differentiated phenotype. Finally, panel (D) depicts a solid, well‐vascularized (V) tumor comprised of sheets of anaplastic tumor cells (6).

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Literature Cited

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Internet Resources
   http://128.249.134.198
  The Greenberg Laboratory home page. Dr. Norman Greenberg can be reached directly at normang@bcm.tmc.edu.
   http://www.jax.org
  The Jackson Laboratory homepage.
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