Generation of Human Acute Lymphoblastic Leukemia Xenografts for Use in Oncology Drug Discovery

Linda Holmfeldt1, Charles G. Mullighan2

1 The Beijer Laboratory for Gene and Neurosciences, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, 2 Pathology, St. Jude Children's Research Hospital, Memphis
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.32
DOI:  10.1002/0471141755.ph1432s68
Online Posting Date:  March, 2015
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Abstract

The establishment of reproducible mouse models of acute lymphoblastic leukemia (ALL) is necessary to provide in vivo therapeutic test systems that recapitulate human ALL, and for amplification of limited amounts of primary tumor material. A popular assay is the primary xenograft model that utilizes immunocompromised mice. The protocol includes injection of primary patient tumor specimens into mice with subsequent serial passaging of the tumors by retransplants of cells harvested from the mouse bone marrow and spleen. The tumors generated are then used for genomic profiling, ex vivo compound testing, mechanistic studies and retransplantation. Detailed in this unit are procedures for the establishment and maintenance of primary ALL xenograft panels for use in basic research and translational studies. © 2015 by John Wiley & Sons, Inc.

Keywords: xenograft; acute lymphoblastic leukemia; mouse model

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transplantation of Primary Leukemia Cells into Immunocompromised Mice
  • Support Protocol 1: Density Gradient Centrifugation
  • Support Protocol 2: Enrichment of Human CD19‐Positive Cells
  • Support Protocol 3: Purification of Primary ALL Cells by Fluorescence‐Activated Cell Sorting (FACS)
  • Support Protocol 4: FACS Analysis of Peripheral Blood, Bone Marrow, and Spleen Cells from Xenografted Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transplantation of Primary Leukemia Cells into Immunocompromised Mice

  Materials
  • 8‐ to 12‐week‐old NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laboratory)
  • Primary patient sample(s) or tumor cell line(s)
  • Dry ice
  • Medium for cell thawing: Iscove's Modified Dulbecco's Medium (IMDM; Gibco, cat. no. 12440)
  • Sterile fetal bovine serum (FBS; e.g., Sigma‐Aldrich, cat. no. F0926)
  • Penicillin‐Streptomycin‐Glutamine (Gibco, cat. no. 10378‐016)
  • Optional: DNase I from bovine pancreas (Sigma‐Aldrich, cat. no. D4263)
  • Optional: Hanks’ Balanced Salt Solution (Gibco, cat. no. 14185‐052)
  • Optional: Anticoagulant citrate‐dextrose solution A (Sigma‐Aldrich, cat. no. C3821)
  • Optional: HEPES (Sigma‐Aldrich, cat. no. H4034)
  • Phosphate‐buffered saline (PBS; Lonza, cat. no. 17‐516 F)
  • 3% Acetic Acid with Methylene Blue (StemCell Technologies, cat. no. 07060)
  • 70% (v/v) Ethanol
  • FITC‐conjugated human CD45 antibodies (Becton Dickinson, cat. no. 340664)
  • PE‐conjugated human CD19 antibodies (Becton Dickinson, cat. no. 349209)
  • APC‐conjugated human CD7 antibodies (Life Technologies, cat. no. MHCD0705)
  • PE‐Cy7‐conjugated mouse CD45 antibodies (Becton Dickinson, cat. no. 552848)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI; e.g., Gibco, cat. No. D1306)
  • Tagging system (e.g., ear tagging, toe clipping, or chip implants)
  • Possible access to a cesium irradiator or equivalent
  • 37°C water bath
  • 15‐ml conical centrifuge tubes (Becton Dickinson, cat. no. 352097)
  • Centrifuge
  • Hemacytometer
  • 5‐ml round‐bottom tube with filter cap (Corning, cat. no. 352235)
  • Biosafety cabinet
  • Heating lamp
  • 1‐ml syringes (Becton Dickinson, cat. no. 309659)
  • 27‐G ½‐in. needle (Becton Dickinson, cat. no. 305109)
  • Mouse restrainer (see Fig.  for an example; an inner diameter of 25 mm is usually appropriate for NSG mice at 8‐ to 12‐weeks‐old)
  • All‐purpose sponges (e.g., Kendall Healthcare, cat. no. 9022)
  • Fluorescence‐activated cell sorter
  • Scissors or razor blades
  • 70‐μm cell strainer (Fisher, cat. no. 22‐363‐548)
  • 25‐G 5/8‐in. needle (Becton Dickinson, cat. no. 305122)
  • 10‐ml syringes (Becton Dickinson, cat. no. 309604)

Support Protocol 1: Density Gradient Centrifugation

  Materials
  • Cell suspension (e.g., bone marrow aspirate or peripheral blood; see the protocol 1Basic Protocol)
  • Hanks’ Balanced Salt Solution (Gibco, cat. no. 14185‐052)
  • HEPES (Sigma Aldrich, cat. no. H4034)
  • Heparin (Sigma‐Aldrich, cat. no. H3149)
  • Accu‐Prep, Lymphocytes (Accurate Chemical and Scientific Corp, cat. no. AN5565)
  • Biosafety cabinet
  • 50‐ml conical test tubes (Fisher Scientific, cat. no. 2098)
  • 15‐ml conical centrifuge tubes (Becton Dickinson, cat. no. 352097)
  • Transfer pipets, sterile (Fisher Scientific, cat. no. 13‐711‐20)

Support Protocol 2: Enrichment of Human CD19‐Positive Cells

  Materials
  • Dynabeads CD19 Pan B (Life Technologies, cat. no. 11143D)
  • Isolation buffer (see recipe)
  • Iscove's Modified Dulbecco's Medium (IMDM; Gibco, cat. no. 12440)
  • Sterile fetal bovine serum (FBS; e.g., Sigma‐Aldrich, cat. no. F0926)
  • Penicillin‐Streptomycin‐Glutamine (Gibco, cat. no. 10378‐016)
  • DETACHaBEAD CD19 (Life Technologies, cat. no. 12506D)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Lonza, cat. no. 17‐516 F)
  • 0.4% Trypan Blue Solution (Gibco, cat. no. 15250‐061)
  • Vortex mixer
  • 15‐ml conical centrifuge tubes (Becton Dickinson, cat. no. 352097)
  • Magnet (e.g., DynaMag‐15 Magnet, Life Technologies, cat. no. 12301D)
  • MACSmix Tube Rotator (Miltenyi Biotec, cat. no. 130‐090‐753)
  • Hemacytometer

Support Protocol 3: Purification of Primary ALL Cells by Fluorescence‐Activated Cell Sorting (FACS)

  Materials
  • Primary patient sample(s) or tumor cell line(s)
  • Iscove's Modified Dulbecco's Medium (IMDM; Gibco, cat. no. 12440)
  • Sterile fetal bovine serum (FBS; e.g., Sigma‐Aldrich, cat. no. F0926)
  • Penicillin‐Streptomycin‐Glutamine (Gibco, cat. no. 10378‐016)
  • Normal Rabbit Serum (Life Technologies, cat. no. 10510)
  • FITC conjugated human CD45 antibodies (Becton Dickinson, cat. no. 340664)
  • PE conjugated human CD19 antibodies (Becton Dickinson, cat. no. 349209)
  • APC conjugated human CD7 antibodies (Life Technologies, cat. no. MHCD0705)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI; e.g., Gibco, cat. No. D1306)
  • Ice

Support Protocol 4: FACS Analysis of Peripheral Blood, Bone Marrow, and Spleen Cells from Xenografted Mice

  Materials
  • Cell suspension
  • Phosphate‐buffered saline (PBS; Lonza, cat. no. 17‐516 F)
  • Sterile fetal bovine serum (FBS; e.g., Sigma‐Aldrich, cat. no. F0926)
  • CD16/CD32 (Becton Dickinson, cat. no. 553142)
  • FITC‐conjugated human CD45 antibodies (Becton Dickinson, cat. no. 340664)
  • PE‐conjugated human CD19 antibodies (Becton Dickinson, cat. no. 349209)
  • APC‐conjugated human CD7 antibodies (Life Technologies, cat. no. MHCD0705)
  • PE‐Cy7 conjugated mouse CD45 antibodies (Becton Dickinson, cat. no. 552848)
  • Ice
  • 4′,6‐diamidino‐2‐phenylindole (DAPI; e.g., Gibco, cat. no. D1306)
  • BD Calibrite Beads (Unlabeled, FITC and PE beads; Becton Dickinson, cat. no. 349502)
  • BD Calibrite APC Beads (Becton Dickinson, cat. no. 340487)
  • Lysis machine, e.g., BD Lyse Wash Assistant (Becton Dickinson, cat. no. 337146)
  • 5‐ml round‐bottom tube with filter cap (Corning, cat. no. 352235)
  • Vortex mixer
  • Flow cytometer
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Figures

Videos

Literature Cited

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