Preclinical Chemotherapeutic Tumor Models of Common Childhood Cancers: Solid Tumors, Acute Lymphoblastic Leukemia, and Disseminated Neuroblastoma

Christopher L. Morton1, Rachel A. Papa2, Richard B. Lock2, Peter J. Houghton1

1 St. Jude Children's Research Hospital, Memphis, Tennessee, 2 Children's Cancer Institute Australia for Medical Research, Randwick, NSW, Australia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.8
DOI:  10.1002/0471141755.ph1408s39
Online Posting Date:  December, 2007
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Abstract

This unit presents three models used in the Pediatric Preclinical Testing Program of the National Cancer Institute (NCI) for preclinical testing of new chemical entities (NCEs), along with appropriate methods for data analysis. The first is the classical subcutaneous xenograft model used for many solid tumors, the second is the disseminated human leukemia model established by Lock and colleagues, and the third is a disseminated model of neuroblastoma that recapitulates many of the characteristics of advanced clinical disease. Curr. Protoc. Pharmacol. 39:14.8.1‐14.8.21. © 2007 by John Wiley & Sons, Inc.

Keywords: preclinical testing; developmental therapeutics; xenograft models

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preclinical Drug Testing Using Subcutaneous Human Xenografts in SCID Mice
  • Basic Protocol 2: Preclinical Drug Testing Using Acute Lymphoblastic Leukemia (ALL) Xenograft Models of Systemic Disease
  • Basic Protocol 3: Preclinical Chemotherapy Testing Using a Disseminated Model of Neuroblastoma
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preclinical Drug Testing Using Subcutaneous Human Xenografts in SCID Mice

  Materials
  • A‐33 disinfectant (Ecolab Professional Products, http://www.ecolab.com, cat. no. 61122362)
  • 100× penicillin/streptomycin solution (Sigma, cat. no. P4333)
  • Medium 199 with Earle's salts, L‐glutamine, and sodium bicarbonate (Invitrogen, cat. no. 11150‐059)
  • Tumor tissue: tumor biopsy material obtained as rapidly as possible from operating room or through pathology laboratory after appropriate Institutional Review Board (IRB) approval
  • Isoflurane, USP (Baxter) and oxygen source (see manufacturer's instructions for anesthesia apparatus
  • CB17SC‐M‐F scid−/− mice 6–10 weeks old (Taconic Farms, cat. no. CB17SC‐M), maintained under barrier conditions (filter‐top cages, only opened in HEPA‐filtered containment)
  • 70% (v/v) ethanol
  • Vetbond tissue adhesive (Webster Veterinary Supply, cat. no. 480200)
  • Tumor cell line growing in monolayer culture and appropriate serum‐containing medium
  • Chemotherapeutic agent (new chemical entity; NCE) to be tested
  • Anesthesia machine/chamber (VetEquip, http://www.vetequip.com) with nose cone appropriate for mice
  • Gauze
  • Sterile dissecting instruments (scalpel, scissors, forceps)
  • Sterile 100‐mm petri dishes
  • Rodent ear tags
  • Coulter Counter (Beckman Coulter) or hemacytometer
  • Centrifuge
  • 1‐cc tuberculin syringes
  • 21‐G needles
  • Animal hair trimmers
  • Digital calipers (Ultra‐CAL IV, FV Fowler Co.)
CAUTION: All transplant procedures must be performed in a Class 2 (Type A/B3) biological safety cabinet. All procedures for handling surgical biopsy tissue should assume potential contamination of the material with human pathogens.

Basic Protocol 2: Preclinical Drug Testing Using Acute Lymphoblastic Leukemia (ALL) Xenograft Models of Systemic Disease

  Materials
  • 6‐ to 8‐week‐old female NOD/SCID (NOD/LtSz‐scid/scid) mice (The Jackson Laboratory)
  • Fresh or cryopreserved mononuclear cell preparations from bone marrow or peripheral blood biopsies from individuals with acute lymphoblastic leukemia (IRB approval will be required to obtain this material)
  • Antibodies (available from various suppliers):
    • Fluorescein isothiocyanate (FITC)–conjugated anti‐mouse CD45
    • Allophycocyanin (APC)–conjugated anti–human CD45 (leukocyte common antigen, Ly‐5)
    • APC‐conjugated anti–human CD19
    • APC‐conjugated anti–human CD7
    • FITC‐ and APC‐conjugated anti‐murine IgG isotype control antibodies (store at 4°C in the dark)
  • Complete RPMI medium/10% FBS (see recipe)
  • Trypan blue
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (CMF‐PBS; e.g., Invitrogen)
  • Flow cytometry buffer (see recipe)
  • RBC (red blood cell) lysis solution (see recipe)
  • Normal mouse serum
  • Density gradient lymphocyte isolation medium (polysucrose–sodium metrizoate): e.g., Ficoll‐Paque, Lymphoprep, or Histopaque
  • Freeze‐down solution (see recipe)
  • Liquid nitrogen
  • Chemotherapeutic agent (new chemical entity; NCE) to be tested
  • ∼21 × 21 × 6–cm Perspex box (Fig. )
  • “Phantom” mice: 25‐g, 7 × 3 × 2–cm autoclavable plastic blocks, sterile
  • Sterile cotton surgical drape or sheet, ∼1 m2
  • γ‐radiation source (linear accelerator instrument, e.g., PRIMUS or ONCOR Impression from Siemens)
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Hemacytometer
  • 1‐ml insulin syringes with 27‐G, 1/2‐in. needles
  • High‐power (>150 W) infrared heat lamp (e.g., Philips Electronics, http://www.philips.com)
  • ∼20 × 20 × 18–cm Perspex mouse restrainer (Fig. )
  • 23‐G, 1–1/4‐in. needles
  • Commercial mini–blood collection tubes containing EDTA or heparin
  • Scissors and forceps, sterile
  • Flow cytometry tubes
  • Multiparameter flow cytometer (530 nm and 670 nm; Becton Dickinson instrumentation is recommended, various models and software are available, and all are suitable)
  • Harvest tubes: 1.5‐ml microcentrifuge tubes containing 1 ml complete medium
  • Autoclaved stainless steel tea strainer
  • 40‐µm cell strainers (BD Biosciences)
  • 50‐ml centrifuge tubes
  • 10‐ and 20‐ml syringes
  • Mixing cannula (12.5‐cm polypropylene mixing cannula with luer adapter; Unomedical, http://www.unomedical.com)
  • 1‐ml cryovials
  • Cryo 1°C Freezing Containers (Mr. Frosty from Nalgene)
  • Dewar flasks and other equipment for liquid nitrogen storage
NOTE: Conduct all procedures involving these mice in a Class II biological safety cabinet.NOTE: The appropriate dosage for all irradiation procedures should be determined by radiologists to ensure its accuracy.

Basic Protocol 3: Preclinical Chemotherapy Testing Using a Disseminated Model of Neuroblastoma

  Materials
  • 0.05% trypsin‐EDTA (Mediatech, cat. no. 25–051‐CI)
  • Neuroblastoma cell line NB‐1691 or other neuroblastoma cell line of interest (Children's Oncology Group; http://www.childrensoncologygroup.org)
  • Complete RPMI medium/10% FBS (see recipe), without penicillin or streptomycin
  • CB17SC‐M‐F scid−/− mice, 6‐ to 10‐weeks‐old (Taconic Farms, cat. no. CB17SC‐M), maintained under barrier conditions (filter top cages, only opened in HEPA‐filtered containment)
  • Chemotherapeutic agent (new chemical entity; NCE) to be tested
  • Coulter Counter (Beckman Coulter) or hemacytometer
  • Centrifuge
  • 1‐cc tuberculin syringes
  • 25‐G needles
  • Additional reagents and equipment for xenograft implantation and determination of chemotherapeutic efficacy ( protocol 2)
NOTE: All procedures must be performed in a Class 2 (Type A/B3) biological safety cabinet.
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Figures

Videos

Literature Cited

Literature Cited
   American Veterinary Medical Association Panel on Euthanasia. 2000. Report of the American Veterinary Medical Association Panel on Euthanasia. J. Am. Vet. Med. Assoc. 218:669‐696.
   Bellet, R.E., Danna, V., Mastrangelo, M.J., and Berd, D. 1979. Evaluation of a “nude” mouse‐human tumor panel as a predictive secondary screen for cancer chemotherapeutic agents. J. Natl. Cancer Inst. 63:1185‐1188.
   Bosma, M.J.and Carroll, A.M. 1991. The SCID mouse mutant: definition, characterization, and potential uses. Annu. Rev. Immunol. 9:323‐350.
   Boven, E., Winograd, B., Fodstad, O., Lobbezoo, M.W., and Pinedo, H.M. 1988. Preclinical phase II studies in human tumor lines: A European multicenter study. Eur. J. Cancer Clin. Oncol. 24:567‐573.
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   Houghton, J.A. and Taylor, D.M. 1978. Maintenance of biological and biochemical characteristics of human colorectal tumours during serial passage in immune‐deprived mice. Br. J. Cancer 37:199‐212.
   Houghton, P.J., Morton, C.L., Tucker, C., Payne, D., Favours, E., Cole, C., Gorlick, R., Kolb, E.A., Zhang, W., Lock, R., Carol, H., Tajbakhsh, M., Reynolds, C.P., Maris, J.M., Courtright, J., Keir, S.T., Friedman, H.S., Stopford, C., Zeidner, J., Wu, J., Liu, T., Billups, C.A., Khan, J., Ansher, S., Zhang, J., and Smith, M.A. 2007a. The pediatric preclinical testing program: Description of models and early testing results. Pediatr. Blood Cancer. In press.
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   Maris, J.M., Courtright, J., Houghton, P.J., Morton, C.L., Gorlick, R., Kolb, E.A., Lock, R., Tajbakhsh, M., Reynolds, C.P., Keir, S.T., Wu, J., and Smith, M.A. 2007. Initial testing of the VEGFR inhibitor AZD2171 by the pediatric preclinical testing program. Pediatr. Blood Cancer. In press.
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   Tajbakhsh, M., Houghton, P.J., Morton, C.L., Kolb, E.A., Gorlick, R., Maris, J.M., Keir, S.T., Wu, J., Reynolds, C.P., Smith, M.A., and Lock, R.B. 2007. Initial testing of cisplatin by the pediatric preclinical testing program. Pediatr. Blood Cancer. In press.
   Thompson, J., Guichard, S.M., Cheshire, P.J., Richmond, L.B., Poquette, C.A., Ragsdale, S.T., Webber, B., Lorsbach, R., Danks, M.K., and Houghton, P.J. 2001. Development, characterization and therapy of a disseminated model of childhood neuroblastoma in SCID mice. Cancer Chemother. Pharmacol. 47:211‐221.
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