Animal Models of Multiple Myeloma and Their Utility in Drug Discovery

Richard A. Campbell1, James R. Berenson1

1 Institute for Myeloma & Bone Cancer Research, West Hollywood, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.9
DOI:  10.1002/0471141755.ph1409s40
Online Posting Date:  March, 2008
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Abstract

To evaluate potential new therapies and targets for treating multiple myeloma (MM), reproducible, biologically relevant in vivo models are required. Preclinical in vivo models of human MM allow investigators to evaluate novel therapies alone and in combination and quickly translate these results to the clinic where patients directly benefit, whether in the form of a new clinical trial, new doses and schedules, or new drug combinations. Presented in this unit are protocols for generating and maintaining a human extramedullary MM tumor in mice. Additionally, the extramedullary tumor can be excised and digested into a single‐cell suspension and the human MM cells injected into mice subcutaneously, intravenously, or intratibially. Once these tumors are generated, they can be used to evaluate novel anti‐MM agents and other therapies. Curr. Protoc. Pharmacol. 40:14.9.1‐14.9.22. © 2008 by John Wiley & Sons, Inc.

Keywords: multiple myeloma; animal model; SCID mice; xenograft; preclinical studies

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generating Primary Human Multiple Myeloma Tumors
  • Basic Protocol 2: Maintaining Primary Multiple Myeloma Implants In Vivo Through Serial Intramuscular Passages in SCID Mice
  • Basic Protocol 3: Generating a Single‐Cell Suspension from a Growing Extramedullary Multiple Myeloma Tumor
  • Basic Protocol 4: Using the Generated Intramuscular Tumors to Evaluate Anti‐Myeloma Agents
  • Support Protocol 1: Retro‐Orbital Blood Collection and Human Immunoglobulin Detection
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generating Primary Human Multiple Myeloma Tumors

  Materials
  • 6‐ to 8‐week‐old male CB17 SCID mice (Charles River Laboratories)
  • Human multiple myeloma patient whole‐core bone marrow biopsy transported at room temperature in a sterile specimen container with sterile RPMI 1640 supplemented with 10% FBS; the biopsy material must be obtained from a research center or other facility that specializes in the treatment of multiple myeloma
  • RPMI 1640 supplemented with 10% fetal bovine serum (FBS)
  • 12.5 mg/ml ketamine hydrochloride in 0.9% NaCl
  • 1.25 mg/ml xylazine in 0.9% NaCl
  • Isoflurane
  • Ophthalmic ointment (Vetropolycin, Pharmaderm)
  • 0.9% (w/v) NaCl (USP for injection), warm
  • 0.5 mg/ml buprenorphine in 0.9% NaCl
  • Anti–asialo GM1 rabbit serum (Wako, cat. no. 986‐10001)
  • γ‐irradiator
  • Petri dishes, sterile
  • Surgical equipment including:
    • Sterile hemostatic forceps
    • Dressing forceps
    • Half‐curved dressing forceps
    • Tissue forceps
    • Straight 15‐mm blade scissors
    • Curved 15‐mm blade scissors
    • Knife handle no. 3 fitting no. 11 scalpel blade
    • No.11 scalpel blade
    • Coated Vicryl sutures (Ethicon)
    • Surgical gauze
    • Autoclip 9 mm wound clips (Becton Dickinson)
    • Wound clip applicator
  • Animal clippers
  • Precision vaporizer and nose cone for administering isoflurane (available at institutional animal care facility)
  • Operating board
  • Transpore surgical tape
  • Povidone‐iodine scrub swabsticks
  • 70% sterile isopropyl alcohol pads
  • Syringes and 29‐G needles
  • Calipers for tumor measurement
  • Additional reagents and equipment for anesthesia of mice (Donovan and Brown, ), parenteral injection of mice (Donovan and Brown, ), and blood collection from mice and determination of paraprotein levels ( protocol 5)

Basic Protocol 2: Maintaining Primary Multiple Myeloma Implants In Vivo Through Serial Intramuscular Passages in SCID Mice

  Materials
  • Donor mouse bearing tumor (from protocol 1)
  • RPMI 1640 supplemented with 10% fetal bovine serum (FBS)
  • 12.5 mg/ml ketamine hydrochloride in 0.9% NaCl
  • 1.25 mg/ml xylazine in 0.9% NaCl
  • γ irradiator
  • Petri dish, sterile
  • Surgical equipment including:
    • Sterile hemostatic forceps
    • Dressing forceps
    • Half‐curved dressing forceps
    • Tissue forceps
    • Straight 15‐mm blade scissors
    • Curved 15‐mm blade scissors
    • Knife handle no. 3 fitting no. 11 scalpel blade
    • No.11 scalpel blade
    • Coated Vicryl sutures (Ethicon)
    • Surgical gauze
    • Autoclip 9‐mm wound clips (Becton Dickinson)
    • Wound clip applicator
  • Animal clippers
  • Operating board
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, ), anesthesia of mice (Donovan and Brown, ), and implantation of tumors ( protocol 1, steps 6 to 18)

Basic Protocol 3: Generating a Single‐Cell Suspension from a Growing Extramedullary Multiple Myeloma Tumor

  Materials
  • RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS)
  • Donor mouse bearing tumor (from protocol 1)
  • Pronase E (EMD Bioscience; http://www.emdbiosciences.com)
  • Naïve 6‐ to 8‐week‐old CD17 SCID mice (Charles River Laboratories) irradiated 24 hr prior to implantation with 250 cGy of γ radiation
  • Sterile Petri dish
  • Sterile scissors
  • Sterile forceps
  • Sterile 10‐ml and 1‐ml pipets
  • Sterile 50‐ml conical centrifuge tube
  • Centrifuge
  • Sterile 0.22‐µm pore‐size 50‐ml filter‐top conical tube
  • Platform rocker
  • Sterile 200‐µm nylon mesh filters, cut into 4 × 4–cm pieces
  • Additional reagents and equipment for euthanasia (Donovan and Brown, ) and parenteral injection of mice (Donovan and Brown, )

Basic Protocol 4: Using the Generated Intramuscular Tumors to Evaluate Anti‐Myeloma Agents

  Materials
  • Donor mouse bearing tumor (from protocol 1)
  • RPMI 1640 supplemented with 10% fetal bovine serum (FBS)
  • 12.5 mg/ml ketamine hydrochloride in 0.9% NaCl
  • 1.25 mg/ml xylazine in 0.9% NaCl
  • Arsenic trioxide (ATO; Cephalon)
  • Bortezomib (Millennium Pharmaceuticals)
  • Melphalan (Sigma‐Aldrich)
  • γ irradiator
  • Petri dish, sterile
  • Surgical equipment including:
    • Sterile hemostatic forceps
    • Dressing forceps
    • Half‐curved dressing forceps
    • Tissue forceps
    • Straight 15‐mm blade scissors
    • Curved 15‐mm blade scissors
    • Knife handle No. 3 fitting no. 11 scalpel blade
    • No.11 scalpel blade
    • Coated Vicryl sutures (Ethicon)
    • Surgical gauze
    • Autoclip 9‐mm wound clips (Becton Dickinson)
    • Wound clip applicator
  • Animal clippers
  • Operating board
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, ), anesthesia of mice (Donovan and Brown, ), implantation of tumors ( protocol 1, steps 6 to 18), blood collection from mice and determination of paraprotein levels ( protocol 5), and parenteral injection of mice (Donovan and Brown, )

Support Protocol 1: Retro‐Orbital Blood Collection and Human Immunoglobulin Detection

  Materials
  • Mouse
  • Isoflurane
  • Precision vaporizer and nose cone for administering isoflurane (available at institutional animal care facility)
  • Heparinized microhematocrit capillary tubes (Fisher)
  • 0.6‐ml microcentrifuge tube
  • Microcentrifuge
  • Human immunoglobulin ELISA kit (Bethyl Laboratories; http://www.bethyl.com)
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Figures

Videos

Literature Cited

Literature Cited
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