Preclinical Orthotopic and Intracardiac Injection Models of Human Breast Cancer Metastasis to Bone and Their Use in Drug Discovery

Ellen Scepansky1, Robert Goldstein2, Michael Rosenblatt3

1 Tufts Medical Center, Division of Hematology/Oncology, Boston, Massachusetts, 2 Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, Massachusetts, 3 Tufts University School of Medicine, Department of Physiology and Medicine, Boston, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.18
DOI:  10.1002/0471141755.ph1418s52
Online Posting Date:  March, 2011
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Abstract

Breast cancer is the most common malignancy in women, with the development of distant metastases rendering the condition incurable. Relatively little is known about the factors governing the progression from primary tumor to metastasis, in part because of the difficulty in modeling what is a complex series of events. Detailed in this unit are descriptions of two murine models of breast cancer metastasis to bone that can be used to screen the effectiveness of new chemical entities on this disease process. Curr. Protoc. Pharmacol. 52:14.18.1‐14.18.23. © 2011 by John Wiley & Sons, Inc.

Keywords: breast cancer; bone metastasis; orthotopic model; intracardiac model; bioluminescent imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Subcutaneous Implantation of Human Bone Into Immunocompromised Mice
  • Basic Protocol 2: Orthotopic Implantation of Human Breast Cancer Cells Into Immunocompromised Mice Containing Ectopic Human Bone Tissue
  • Basic Protocol 3: Bioluminescent Imaging of Breast Cancer Lesions in Mice
  • Alternate Protocol 1: Intracardiac Injection of Breast Cancer Cells Into Immunocompromised Mice Containing Human Bone Tissue
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Subcutaneous Implantation of Human Bone Into Immunocompromised Mice

  Materials
  • Human cancellous bone—femoral head(s) from total hip replacement surgery
  • Sterile phosphate‐buffered saline (PBS; Fisher Scientific)
  • 6‐ to 8‐week‐old female NOD/SCID mice (Jackson Laboratories)
  • Betadine surgical scrub
  • Individually packaged 70% alcohol prep pads
  • Buprenorphrine for injection, 0.1 mg/kg per animal (e.g., Alstoe Animal Health; please note that this can only be supplied to a veterinarian)
  • Antibiotic suspension (sulfamethoxazole and trimethoprim 200 mg/40 mg per 5 ml, e.g., brand names Sulfatrim, septra; e.g., California Vet Supply, http://www.calvetsupply.com)
  • Bone cutting saw: tabletop model (Marmed) or hand‐held saw (Styker Instruments) plus clamps to immobilize the sample
  • Bone biopsy trephine cutter (DePuy Mitek) or other equivalent coaxial tool of 5‐ or 6‐mm diameter
  • Sterile towels and gauze
  • Circulating‐water heating pad (part of the anesthesia unit)
  • Anesthesia delivery system (induction chamber, nose cone apparatus, oxygen and isoflurane source, heated surgical stage; e.g., Colonial Medical Supply)
  • Tape (paper surgical or clear office type)
  • Electric clippers
  • Sterile gauze
  • Surgical gloves and masks, sterile
  • Autoclaved surgical instruments: use small‐sized (4.5‐in.) operating room supplies or items specifically manufactured for small‐animal surgery (Roboz) including:
    • Straight scissors
    • Hemostat or needle holder
    • Two pairs of forceps
  • 9‐mm wound clips with matched stapler and removal tool (Becton Dickinson)
  • Ear punch for identification of individual mice (Kent Scientific)
  • Glass‐bead autoclave (for sterilization of instruments between each animal's procedure; e.g., Cole‐Parmer, cat. no. 10779‐05)
  • 0.5‐ or 1.0‐ml syringes equipped with 28‐G needles
  • Animal cages

Basic Protocol 2: Orthotopic Implantation of Human Breast Cancer Cells Into Immunocompromised Mice Containing Ectopic Human Bone Tissue

  Materials
  • Firefly luciferase‐expressing, bone‐avid human breast cancer cell line, either MDA‐MB‐231‐luc2 (Caliper Life Sciences) or SUM 1315 (Asterand) transfected with lentiviral particles containing the luc gene
  • Medium for cell growth: for MDA‐MB‐231 cells, RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/streptomycin; for SUM‐1315 cells, F‐12 (Ham's) supplemented with 5% fetal bovine serum, penicillin/streptomycin, 5 µg/ml insulin, and 10 ng/ml epidermal growth factor (EGF)
  • Phosphate‐buffered saline (PBS)
  • 0.05% (w/v) trypsin‐EDTA (Invitrogen, cat. no. 25200‐056)
  • Matrigel basement membrane matrix (BD Biosciences)
  • Female NOD/SCID mice with bone implants placed 4 weeks earlier (if experimenter plans to examine metastasis to human bone; see protocol 1)
  • Betadine surgical scrub
  • Individually packaged 70% alcohol prep pads
  • Antibiotic suspension (sulfamethoxazole and trimethoprim 200 mg/40 mg per 5 ml, e.g., brand names Sulfatrim, septra; e.g., California Vet Supply, http://www.calvetsupply.com)
  • 150‐mm tissue culture plates
  • 37°C, 5% CO 2 incubator
  • 50‐ml centrifuge tubes
  • Hemacytometer or automated cell counter
  • 2‐ml microcentrifuge tubes
  • Sterile towels and gauze
  • Circulating‐water heating pad (part of the anesthesia unit)
  • Anesthesia delivery system (induction chamber, nose cone apparatus, oxygen and isoflurane source, heated surgical stage)
  • Tape (paper surgical or clear office type)
  • Electric clippers
  • Surgical gloves and mask, sterile
  • Autoclaved surgical instruments: use small‐sized (4.5‐in.) operating room supplies or items specifically manufactured for small‐animal surgery (Roboz) including:
    • Straight scissors
    • Hemostat or needle holder
    • Two pairs of forceps
  • 50‐µl Hamilton syringe with 22‐G needle
  • 9‐mm wound clips with matched stapler and removal tool (Becton‐Dickinson)
  • Glass‐bead autoclave
NOTE: The 4‐week interim period (for the mice bone implants) allows the graft to become fully vascularized while ensuring that the animal is not excessively aged (>9 months) at the end of the experiment. Active hematopoiesis and bone remodeling is present upon histological examination of the implants at the end of a 16‐week experimental period. In general, all implants remain viable, barring immediate post‐operative complications.

Basic Protocol 3: Bioluminescent Imaging of Breast Cancer Lesions in Mice

  Materials
  • Mice bearing tumors expressing luciferase (from protocol 2 or Alternate Protocol)
  • D‐luciferin solution: dissolve powder (e.g., Caliper Life Sciences) in sterile PBS to make concentration 10 mg/ml(aliquots should be protected from light and may be stored indefinitely at −20° to −80°C)
  • 10% buffered formalin
  • 70% ethanol
  • IVIS Imaging Systems (Caliper Life Sciences)
  • Anesthesia delivery system (induction chamber, nose cone apparatus, oxygen and isoflurane source, heated surgical stage; e.g., Colonial Medical Supply)
  • 0.5‐ or 1.0‐ml insulin syringes equipped 29‐G needles
  • Surgical instruments for necropsy: scissors and forceps
  • Petri dishes
  • Additional reagents and equipment for euthanizing the animals (Donovan and Brown, )

Alternate Protocol 1: Intracardiac Injection of Breast Cancer Cells Into Immunocompromised Mice Containing Human Bone Tissue

  Materials
  • Firefly luciferase‐expressing, bone‐avid human breast cancer cell line, e.g., MDA‐MB‐231‐luc2 (Caliper Life Sciences)
  • Medium for cell growth: for MDA‐MB‐231 cells, RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/streptomycin
  • Phosphate‐buffered saline (PBS; Fisher Scientific)
  • 6‐ to 8‐week‐old female NOD/SCID or nude mice (Jackson Laboratory), or mice that have had bone implanted ( protocol 1) four weeks earlier
  • Betadine surgical scrub
  • Individually packaged 70% alcohol prep pads
  • Hemacytometer or automated cell counter
  • Anesthesia delivery system (induction chamber, nose cone apparatus, oxygen and isoflurane source, heated surgical stage; e.g., Colonial Medical Supply)
  • Tape (paper surgical or clear office type)
  • Sterile towels and gauze
  • Surgeon's mask and gloves, sterile
  • 1‐ml insulin syringes with 26‐G needles
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Figures

Videos

Literature Cited

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