Imaging Tumor Cell Movement In Vivo

David Entenberg1, Dmitriy Kedrin1, Jeffrey Wyckoff2, Erik Sahai3, John Condeelis2, Jeffrey E. Segall2

1 These authors contributed equally to this work., 2 Albert Einstein College of Medicine, Bronx, New York, 3 London Research Institute, London, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.7
DOI:  10.1002/0471143030.cb1907s58
Online Posting Date:  March, 2013
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This unit describes the methods that we have been developing for analyzing tumor cell motility in mouse and rat models of breast cancer metastasis. Rodents are commonly used both to provide a mammalian system for studying human tumor cells (as xenografts in immunocompromised mice) as well as for following the development of tumors from a specific tissue type in transgenic lines. The Basic Protocol in this unit describes the standard methods used for generation of mammary tumors and imaging them. Additional protocols for labeling macrophages, blood vessel imaging, and image analysis are also included. Curr. Protoc. Cell Biol. 58:19.7.1‐19.7.19. © 2013 by John Wiley & Sons, Inc.

Keywords: imaging; metastasis; mammary tumor; image analysis

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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation and In Vivo Imaging of Mammary Tumors
  • Support Protocol 1: In Vivo Imaging Microscope Setup
  • Support Protocol 2: Labeling Vasculature and Macrophages
  • Support Protocol 3: Blood Vessel Imaging Using an Indwelling Catheter
  • Support Protocol 4: Second Harmonic Fiber Imaging
  • Basic Protocol 2: Multiphoton Time‐Lapse Image Analysis Using ImageJ and Custom Plugins
  • Support Protocol 5: Separation of Spectral Overlap
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Generation and In Vivo Imaging of Mammary Tumors

  • MTLn3 cells (available from authors; )
  • MTLn3 growth medium (see recipe)
  • 2 mM EDTA (Invitrogen, cat. no. 15575‐038) in calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Invitrogen; also see appendix 2A)
  • Dulbecco's phosphate‐buffered saline (with calcium and magnesium; DPBS; Invitrogen; also see appendix 2A)
  • Female 5‐ to 6‐week‐old BALB/c SCID/Ncr mice (National Cancer Institute), Fisher 344 rats (The Jackson Laboratory), or transgenic mouse tumor models (e.g., MMTV‐pyMT; The Jackson Laboratory)
  • 70% (v/v) ethanol
  • Isoflurane, USP (AERRANE, Baxter)
  • 15‐cm tissue culture dishes (Falcon)
  • 15‐ml conical polypropylene centrifuge tubes, sterile
  • Cell scrapers
  • Centrifuge (e.g., Sorvall GLC‐1)
  • 1‐ml syringes with 25‐G needles for cell injections
  • Oxygen/anesthesia apparatus (Forane Vaporizer, model 100, from SurgiVet)
  • 100% compressed oxygen tanks to be used in combination with vaporizer
  • Surgical instruments
  • In vivo imaging microscope setup ( protocol 2)
  • Multiphoton apparatus setup ( protocol 5)
  • Surgical tape (optional)
  • Additional reagents and equipment for tissue culture techniques including counting cells (unit 1.1), housing of immunocompromised mice (Donovan and Brown, ), imaging blood vessels ( protocol 3 or 3; optional), inhalant anesthesia of rodents (Donovan and Brown, ), euthanasia of rodents (Donovan and Brown, ), and image analysis and quantitation ( protocol 6)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be carried out in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Support Protocol 1: In Vivo Imaging Microscope Setup

  • Mouse with xenograft‐induced tumor ( protocol 1)
  • 70% (v/v) ethanol
  • 20 mg/ml dye labeled–dextran (70 kDa, Invitrogen) in DPBS (Invitrogen; also see appendix 2A)
  • Mouse restrainer (Donovan and Brown, )
  • 1‐ml syringe with 26‐G needle
  • Superglue
  • Additional reagents and equipment for mouse restraint (Donovan and Brown, )

Support Protocol 2: Labeling Vasculature and Macrophages

  • Mouse with xenograft‐induced tumor ( protocol 1)
  • Heparinized PBS solution (Baxter, cat. no. FKB0953G)
  • Indwelling tail vein catheter (no. MTV‐01, SAI Inc.; http://www.sai‐
  • 1‐ml syringes
  • Waterproof marker (e.g., Sharpie)
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Literature Cited

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