Preclinical Models of Pediatric Solid Tumors (Neuroblastoma) and Their Use in Drug Discovery

Danielle M. Patterson1, Jason M. Shohet1, Eugene S. Kim1

1 Baylor College of Medicine, Texas Children's Hospital, Houston, Texas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.17
DOI:  10.1002/0471141755.ph1417s52
Online Posting Date:  March, 2011
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Neuroblastoma is the most common pediatric abdominal solid tumor. This aggressive embryonal malignancy of neural crest origin has a peak age of onset of 22 months, and accounts for ∼11% of all pediatric cancers and 15% of all pediatric cancer deaths. With current treatment protocols, including high‐dose chemotherapy with autologous stem cell transplantation, radiation, and surgery, ∼80% of high‐risk patients go into remission, although the majority relapse and succumb to therapy‐resistant tumors. Long‐term survival rates (>5 years) are <50%. Mouse models of neuroblastoma provide clinically relevant tools for studying the growth and metastasis of this aggressive malignancy, and for testing the efficacy of potentially novel therapeutics in vivo. This unit describes an orthotopic murine model of neuroblastoma using cultured human cells that closely mimics the clinical condition in terms of the bulky intra‐abdominal tumors and other aspects of metastatic disease. Also described are methods for in vivo imaging and monitoring of tumor growth, and procedures for necropsy and tumor preservation for pathological analysis. Curr. Protoc. Pharmacol. 52:14.17.1‐14.17.18. © 2011 by John Wiley & Sons, Inc.

Keywords: neuroblastoma; xenograft; orthotopic; in vivo imaging; bioluminescent imaging; athymic nude mice

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Table of Contents

  • Introduction
  • Basic Protocol 1: Orthotopic Human Xenograft Model in Nude Mice
  • Basic Protocol 2: Bioluminescent Imaging (BLI) of Neuroblastoma Tumor Xenografts
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Orthotopic Human Xenograft Model in Nude Mice

  • Human neuroblastoma cell culture, e.g., IMR‐32 (ATCC)
  • Cell culture medium: serum‐free medium (e.g., RPMI medium 1640, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen), 1% (v/v) penicillin/streptomycin (100× P/S, Invitrogen; final concentration 100 units/ml penicillin and 100 µg/ml streptomycin), and 1% (v/v) of 100× L‐glutamine (Invitrogen; final concentration 2 mM)
  • Phosphate‐buffered saline (PBS), sterile (Invitrogen): cold and room temperature
  • 0.25% trypsin/EDTA (Invitrogen)
  • Trypan blue (Invitrogen)
  • Surface disinfectant (e.g., Clidox‐S, Pharmacal Research Laboratories)
  • Isoflurane, USP (Baxter)
  • Oxygen source for anesthesia machine (see manufacturer's instructions)
  • Betadine solution
  • 4‐ to 6‐week‐old (20 to 25 g) female athymic Ncr nude mice (Harlan/Taconic), maintained under barrier conditions (pathogen‐free conditions provided by plastic cages with sealed air filters, laminar‐air‐flow units, or laminar‐air‐flow rooms; see National Research Council, )
  • Ophthalmic base ointment (Webster Veterinary Supply)
  • 10% (v/v) formalin
  • 100% ethanol
  • 37°C, 5% CO 2 incubator
  • 75‐cm2 cell culture flasks
  • 15‐ml conical tube, sterile
  • Refrigerated centrifuge
  • Coulter counter (Beckman Coulter) or hemacytometer
  • Class 2 biological safety cabinet with laminar flow hood
  • Disposable surgical towels, sterile and plastic lined (Fisher Scientific)
  • Anesthesia machine/chamber with nose cone appropriate for mice (Surgivet or VetEquip)
  • 100‐mm petri dishes, sterile
  • 5.5‐in. Mayo‐Hegar or similar needle holder (Millennium Surgical or Roboz Surgical Instrument Co.)
  • Surgical light (LED or halogen; Storz Medical), optional
  • 4 × 4–in. sterile gauze sponges (Fisher Scientific)
  • Surgical gloves, sterile and fitted (Fisher Scientific)
  • Surgical instruments, sterile (Millennium Surgical or Roboz Surgical Instrument Co.)
    • Disposable, sterile scalpel blade (#10)
    • 4.5‐in. Iris scissors, straight, sharp ends
    • 4.5‐in. Adson toothed tissue forceps
    • 4.75‐in. Adson forceps or delicate dressing forceps, serrated
  • 27‐G needle (1/2 in. long) with sterile 1‐cc slip‐tip syringe
  • Cotton‐tipped swabs, sterile
  • Polysorb 4‐0 sutures (U.S. Surgical) with RB‐1 tapered needle, one pack per 5 to 10 mice
  • 9‐mm wound clips, with applier and remover (VWR)
  • Rodent ear tags to identify individual mice (National Band and Tag Company)
  • Circulating heated water bath, heating pad, or infrared heating lamp (Fisher Scientific)
  • Scale, capable of accurately weighing 0.001 g
  • Additional reagents and equipment for performing necroscopy (unit 7.1, Basic Protocol 4)

Basic Protocol 2: Bioluminescent Imaging (BLI) of Neuroblastoma Tumor Xenografts

  • Neuroblastoma tumor‐bearing mice ( protocol 1) created using cell lines, e.g., IMR‐32 (ATCC), SH‐SY5Y (ATCC), CHLA‐255, or NGP, NB‐1691, transfected or virally transduced using standard protocols with commercially available vectors, e.g., pGL2‐Control Vector (Promega), to express a luminescent reporter gene; contact for information about obtaining or generating these cell lines
  • Luciferase assay kit (e.g., Promega)
  • 1 g D‐luciferin, firefly, potassium salt (Caliper Life Sciences)
  • 1× Dulbecco's phosphate‐buffered saline without magnesium or calcium (DPBS), sterile (Invitrogen)
  • Isoflurane, USP (Baxter)
  • Oxygen source for anesthesia machine (see manufacturer's instructions)
  • Laminar flow tissue culture hood.
  • 50‐ml plastic tube, sterile (Falcon)
  • Aluminum foil
  • 0.2‐µm vacuum filter system (Corning)
  • 1.2‐ml cryovials (Fisher Scientific)
  • Foil‐covered box
  • Alcohol prep pads (70% isopropyl alcohol)
  • Insulin syringe (1‐ml syringe with 27‐G needle)
  • Anesthesia machine/chamber with nose cone appropriate for mice (Surgivet, or VetEquip, and isoflurane, USP (Baxter) and oxygen source (see manufacturer's instructions)
  • In vivo bioluminescent CCD imaging system (Xenogen IVIS Spectrum, Caliper Life Sciences)
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