Utility and Applications of Orthotopic Models of Human Non‐Small Cell Lung Cancer (NSCLC) for the Evaluation of Novel and Emerging Cancer Therapeutics

Verline Justilien1, Alan P. Fields1

1 Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.27
DOI:  10.1002/0471141755.ph1427s62
Online Posting Date:  October, 2013
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Abstract

Lung cancer is a leading cause of cancer deaths worldwide. Despite advances in chemotherapy, radiation therapy, and surgery, lung cancer continues to have a low 5‐year survival rate, highlighting a dire need for more effective means of prevention, diagnosis, prognosis, and treatment. Mouse models that recapitulate the clinical features of advanced human lung cancer are critical for testing novel therapeutic approaches. This unit describes a highly reproducible, easy‐to‐establish orthotopic murine model of lung cancer, provides methods for in vivo imaging and monitoring of tumor growth, and discusses the usefulness of this model for translational lung cancer research and the development of therapeutic strategies. Curr. Protoc. Pharmacol. 62:14.27.1‐14.27.17. © 2013 by John Wiley & Sons, Inc.

Keywords: non‐small cell lung cancer; orthotopic model; bioluminescent in vivo imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Orthotopic NSCLC Mouse Model
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Orthotopic NSCLC Mouse Model

  Materials
  • H1299 lung carcinoma cell line (ATCC) or other cell line suitable for the generation of lung orthotopic tumors (see Table 14.27.1)
  • Complete cell culture medium: RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin
  • Lipofectamine 2000 reagent (Invitrogen)
  • pGL4.51 vector containing a firefly luciferase gene constitutively driven by a CMV promoter and containing a neomycin resistance cassette (Promega, cat. no. E1320)
  • G418 (Invitrogen)
  • 15 mg/ml D‐luciferin, firefly (Caliper Life Sciences, cat. no. 122796)
  • 0.25% trypsin‐EDTA
  • 0.4% trypan blue
  • 1× phosphate‐buffered saline (PBS; Invitrogen)
  • Matrigel, growth factor reduced (BD)
  • Male athymic nude mice (6 to 8 weeks old; Harlan Laboratories) or other suitable mice
  • Ketamine (90 to 120 mg/kg, Cardinal Health)/xylazine (10 mg/kg, Patterson Veterinary Supply) or other appropriate anesthetic
  • 10% povidone‐iodine (Betadine)
  • Ophthalmic ointment (containing neomycin sulfate, polymyxin B sulfate, and bacitracin zinc) (Patterson Veterinary Supply)
  • Buprenorphine (Cardinal Health)
  • Compound(s) for experimental treatment of mice, and vehicle (for control)
  • Isoflurane (100%) (Abbott)
  • 10% buffered formalin (for paraffin embedding) or OCT freezing medium (for storage in liquid nitrogen)
  • Tissue culture flasks and plates [T150 (150 cm2) flasks, 35‐mm plates]
  • In vivo bioluminescent imaging system including inhalational anesthesia vaporizer system with induction chamber (e.g., Xenogen IVIS Spectrum, Caliper Life Sciences)
  • Living Image software for IVIS Spectrum (Caliper Life Sciences)
  • 50‐ml conical centrifuge tubes
  • Sterile surgical towels
  • Heating pad
  • Ear punch
  • 0.5‐ml syringes and needles (27‐G × ½‐in. length; BD Medical)
  • Sterile gloves
  • Sterile alcohol prep pads (70% isopropyl alcohol)
  • 3/10‐cc insulin syringe with 30‐G needle (BD)
  • Sterile surgical scissors and forceps
  • Sterile cotton‐tipped applicators
  • Dry bead sterilizer
  • Wound‐closing surgical metal clips, stapler, and remover (e.g., AutoClip applier, AutoClip remover, and 9‐mm EZ Clip; BD Medical)
  • Heating lamp
  • 25‐G needle
  • 10‐ml syringes
  • 20‐G feeding needle (Roboz)
  • Additional reagents and equipment for fixation in paraffin ( appendix 3D) or by freezing ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
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