Characterization of Matrix Metalloproteinase Inhibitors: Angiogenesis and Tumor Models

Daniel H. Albert1, Steven K. Davidsen1

1 Abbott Laboratories, Abott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.23
DOI:  10.1002/0471141755.ph0523s13
Online Posting Date:  August, 2001
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Abstract

Since the matrix metalloproteinases (MMPs) have an essential role in the process of tumor growth, invasion and metastasis, small molecule MMP inhibitors have the ability to modulate tumor progression in animals and the potential to be of therapeutic benefit to cancer patients. The antiangiogenic properties of MMP inhibitors can be assessed by the measurement of hemoglobin content of Matrigel plugs containing angiogenic growth factors introduced into the flanks of mice. A flank tumor growth model using B16 murine melanoma cells provides a useful means of determining the antitumor effects of MMP inhibitors as well as correlating efficacy with the concentration of drug in blood.

     
 
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Table of Contents

  • Basic Protocol 1: Matrigel Plug In Vivo Angiogenesis Model
  • Basic Protocol 2: Syngeneic Tumor Growth Model
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Matrigel Plug In Vivo Angiogenesis Model

  Materials
  • Matrigel (phenol red‐free, Becton Dickinson; store frozen up to 9 months at −20°C)
  • 16,000 U/ml heparin (Life Technologies) prepared in PBS (e.g., Life Technologies) containing 0.1% (w/v) bovine serum albumin (store at 4°C)
  • 0.25 µg/ml acidic fibroblast growth factor (aFGF; R&D Systems) in PBS containing 0.1% bovine serum albumin (store frozen in aliquots and thaw each aliquot only once)
  • C57 BL/6 female mice, 22 to 24 g, 6 to 8 weeks old (Charles River)
  • Test compound
  • MMP reference inhibitor ABT‐770 (can be obtained from Abbott Laboratories; contact Dr. Daniel H. Albert; )
  • 90% (v/v) acetic acid
  • Saline (0.9% NaCl in water)
  • Hemoglobin standard (30 mg/dl, Sigma)
  • 5 mg/ml 3,3′,5,5′‐tetramethylbenzidine (TMB) in 90% glacial acetic acid (store up to 2 weeks at 4°C)
  • 0.3% hydrogen peroxide
  • Syringes with 25 ⅜‐G needles
  • Dissecting equipment including scissors and forceps
  • Polytron homogenizer (Brinkmann)
  • 0.25‐ml microcentrifuge tubes
  • 96‐well flat‐bottom microtiter plates
  • Plate reader capable of reading absorbance at or near 600 nm and preferably equipped with data analysis software suitable for kinetic readings (e.g., SPECTRAmax Plus384 with SOFTmax PRO, Molecular Devices)
NOTE: Glacial acetic acid, hydrogen peroxide and TMB can be obtained together in a kit (Hemoglobin, Plasma Chemistry kit) from Sigma.

Basic Protocol 2: Syngeneic Tumor Growth Model

  Materials
  • B16‐F10 cells (ATCC #CRL‐6475)
  • Dulbecco's modified Eagle's medium (DMEM; Life Technologies) containing 10% FBS and 1% penicillin (5000 U/ml)‐streptomycin (5000 µg/ml) (Life Technologies)
  • Versene (Life Technologies)
  • Matrigel (phenol red‐free, Becton Dickinson; store frozen up to 9 months at −20°C)
  • 16,000 U/ml heparin (Life Technologies) prepared in PBS (e.g., Life Technologies) containing 0.1% (w/v) bovine serum albumin (store at 4°C)
  • 0.25 µg/ml acidic fibroblast growth factor (aFGF; R&D Systems) in PBS containing 0.1% bovine serum albumin (store frozen in aliquots and thaw each aliquot only once)
  • C57 BL/6 female mice, 22 to 24 g, 6 to 8 weeks old (Charles River)
  • Test compound
  • MMP reference inhibitor ABT‐770 (can be obtained from Abbott Laboratories; contact Dr. Daniel H. Albert; )
  • Sources of CO 2 and O 2 to produce 60% CO 2/40% O 2 mixture
  • 15% (w/v) tripotassium EDTA in phosphate‐buffered saline (PBS; Life Technologies)
  • 30% (v/v) methanol
  • Ethyl acetate
  • Source of N 2
  • 162‐ or 175‐cm2 tissue culture flasks
  • 15‐cm2 conical centrifuge tubes
  • Syringes with 25 ⅜‐G needles
  • Caliper micrometer
  • Capillary tubes
  • Polytron homogenizer (Brinkmann)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique must be followed accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
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Key Reference
   Passaniti et al., 1992. See above.
  Original description of the Matrigel angiogenesis model.
Internet Resource
  http://www.keystonescientific.com
  Provides access to useful technical bulletins on HPLC techniques.
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