Pre‐Clinical Models of Renal Carcinoma and Their Utility in Drug Development

Brenda C. Salumbides1, Kristin M. Lehet2, Georges Ndikuyeze1, Roberto Pili2

1 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland, 2 Roswell Park Cancer Institute, Buffalo, New York
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.13
DOI:  10.1002/0471141755.ph1413s47
Online Posting Date:  December, 2009
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Significant progress has been made in the treatment of patients with advanced renal cancer. In addition to immunotherapy, there are several potentially distinct therapeutic approaches for targeting molecular pathways. The murine models detailed in this unit are useful for testing rational combination strategies. Moreover, animal models contribute immensely to the understanding of the genetic, epigenetic, and biological aspects of human disease. Compared to humans, rodent models are relatively short‐lived and allow for the facile study of clinically relevant pathologies. Animal models for the study of renal cell carcinoma (RCC) are particularly useful for the development of new drugs for kidney cancer. Included in this unit are several in vivo models that are currently used to evaluate therapeutic approaches to renal cancer therapy and to investigate the pathophysiology of this condition. Included are both murine (RENCA) and renal cell carcinomas in subcutaneous and orthotopic models using tumor cell lines and human tumor tissue. Curr. Protoc. Pharmacol. 47:14.13.1‐14.13.19. © 2009 by John Wiley & Sons, Inc.

Keywords: renal cell carcinoma; metastastic tumor; xenograft model; orthotopic implantation; subcutaneous implantation; angiogenesis; bioluminescent imaging; immunotherapy; combination therapy; retinoids

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Table of Contents

  • Introduction
  • Basic Protocol 1: Orthotopic Model of Murine Renal Adenocarcinoma in Syngeneic BALB/c Mice (RENCA Model)
  • Basic Protocol 2: Use of Orthotopic RENCA Model in Drug Screening
  • Basic Protocol 3: Use of Bioluminescent Imaging to Measure Real‐Time Tumor Growth In the RENCA or RCC Model
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Orthotopic Model of Murine Renal Adenocarcinoma in Syngeneic BALB/c Mice (RENCA Model)

  • BALB/c mice (female, 4‐ to 6‐weeks‐old; NIH, NCI Fredrick, or Charles River Laboratories)
  • RENCA cells (ATCC #CRL‐2947)
  • RPMI 1640 supplemented with 1% L‐glutamine, 1% penicillin/streptomycin (Invitrogen) and 10% fetal calf serum
  • Phosphate‐buffered saline (PBS), pH 7.4 (Invitrogen)
  • 1× trypsin/EDTA (Invitrogen)
  • Hanks' balanced salt solution (HBSS; Invitrogen)
  • Trypan blue stain (Sigma‐Aldrich)
  • Ketamine/xylazine mixture (see recipe)
  • Sterile surgical alcohol prep pads, medium (VWR) or 70% ethanol spray
  • Betadine solution swabstick (Fisher Scientific)
  • 25‐, 75‐, and 150‐cm2 tissue culture flasks
  • 15‐ml conical tubes
  • Hemacytometer or automated cell counter
  • 1‐ml syringes and 27‐G needles
  • Balance accurate to 0.1 g
  • Surgical instruments (Fine Science Tools) including (two sets of instruments recommended):
    • Microdissecting scissors
    • 5.5‐in. operating scissors, sharp point
    • 4.5‐in. iris scissors, straight, sharp ends
    • 4.75‐in. curved forceps or delicate dressing forceps, serrated
    • 5.5‐in. Mayo Hegar needle holder (for suturing)
  • Sterile gauze
  • 100‐µl Hamilton syringe with removable needle (1710 NR 100 µl; 22S/61/3 Ecom, Hamilton Company)
  • Microscope
  • Absorbable surgical sutures (Cardinal Health) vicryl 4/0 (1.5 metric), for closing peritoneal layer
  • 9‐mm wound clips (Fisher), with applier and remover
  • Ear punch device (Harvard Apparatus)
  • Heat lamp
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile and sterile technique employed when handling the material. All work is performed in a laminar flow hood to maintain a sterile environment. All individuals handling animals must wear sterile gloves and use sterile instruments. Animals should be anesthesized with an i.p. injection of ketamine/xylazine mixture.

Basic Protocol 2: Use of Orthotopic RENCA Model in Drug Screening

  • Mice orthotopically injected with RENCA cells in subcapsular space of kidney (see protocol 1)
  • CO 2 source
  • H&E stain (Sigma‐Aldrich)
  • Zinc fixative (see recipe)
  • 10% neutral buffered formalin (Sigma‐Aldrich)
  • Liquid nitrogen
  • Bouin's toxin (Ricca Chemical Company, cat. no. 1120‐1)
  • 0.1 M Tris⋅Cl buffer, pH 7.4
  • Animal balance
  • 1‐ml syringes and 27‐G needles
  • Serrated forceps or thread
  • 50‐ml conical tubes
  • Caliper

Basic Protocol 3: Use of Bioluminescent Imaging to Measure Real‐Time Tumor Growth In the RENCA or RCC Model

  • RENCA (ATCC #CRL‐2947) or human RCC cell lines (NCI Frederick)
  • RPMI complete medium (see recipe)
  • Cignal Lenti reporter (SABiosciences)
  • 8 mg/ml polybrene (Sigma Aldrich)
  • Trypsin
  • 25 mg/ml D‐luciferin (CaliperLife Sciences)
  • XTT cell proliferation kit (Roche) or MTT cell proliferation kit (Promega)
  • Test compounds
  • Luciferase‐transfected cells (with modified firefly luciferase gene under control of TRE promoter) (Promega)
  • Inbred BALB/c mice for RENCA (NIH), SCID, or athymic nude mice for RCC (NIH)
  • 6‐ and 96‐well plates
  • Bioluminescence imager (e.g., Xenogen IVIS camera system)
  • Spectrophotometer
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