Patient‐Derived Models of Human Breast Cancer: Protocols for In Vitro and In Vivo Applications in Tumor Biology and Translational Medicine

Yoko S. DeRose1, Keith M. Gligorich1, Guoying Wang2, Ann Georgelas3, Paulette Bowman3, Samir J. Courdy4, Alana L. Welm2, Bryan E. Welm5

1 These authors contributed equally to this work., 2 Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, 3 Tissue Resource and Applications Core Shared Resource Facility, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, 4 Research Informatics, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, 5 Department of Surgery, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.23
DOI:  10.1002/0471141755.ph1423s60
Online Posting Date:  March, 2013
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Abstract

Research models that replicate the diverse genetic and molecular landscape of breast cancer are critical for developing the next‐generation therapeutic entities that can target specific cancer subtypes. Patient‐derived tumorgrafts, generated by transplanting primary human tumor samples into immune‐compromised mice, are a valuable method to model the clinical diversity of breast cancer in mice, and are a potential resource in personalized medicine. Primary tumorgrafts also enable in vivo testing of therapeutics and make possible the use of patient cancer tissue for in vitro screens. Described in this unit are a variety of protocols including tissue collection, biospecimen tracking, tissue processing, transplantation, and three‐dimensional culturing of xenografted tissue, which enable use of bona fide uncultured human tissue in designing and validating cancer therapies. Curr. Protoc. Pharmacol. 60:14.23.1‐14.23.43. © 2013 by John Wiley & Sons, Inc.

Keywords: breast cancer; tumorgraft; organoid; 3D culture; Matrigel; EHS matrix; biospecimen; tissue repository; tissue collection; xenograft

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Processing Human Breast Tumors and Tumorgrafts into Fragments, Organoids, and Single Cells
  • Basic Protocol 2: Processing Human Body Fluid (e.g., Pleural Effusion, Ascites)
  • Basic Protocol 3: Characterization of Single Cells Isolated from Human Tumors, Body Fluid, or from Tumor Graft Tissue Using Fluorescence Activated Cell Sorting (FACS)
  • Basic Protocol 4: Subcutaneous Implantation of Estrogen Pellets in Mice Receiving Estrogen Receptor–Positive Tumors
  • Basic Protocol 5: Implantation of Breast Tumor Tissue Fragments into Mice
  • Basic Protocol 6: Transplanting Tumor Cells and Organoids into Mice
  • Basic Protocol 7: Harvesting Tumorgrafts from Mice
  • Basic Protocol 8: Producing and Utilization of Lentiviruses for Transduction of Tumor Cells
  • Basic Protocol 9: Three‐Dimensional Primary Tumor Cultures
  • Support Protocol 1: Obtaining Fresh Breast Tumor (e.g., Surgical Specimens, Body Fluids, and Metastases) from Patients
  • Support Protocol 2: Preparation of Estrogen Pellets
  • Support Protocol 3: Preparation of Growth Factor–Reduced EHS Matrix
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Processing Human Breast Tumors and Tumorgrafts into Fragments, Organoids, and Single Cells

  Materials
  • Human breast primary tumor tissue, metastasis tissue, effusion samples (fresh; the quantity usually varies) or fresh tumorgrafts derived from mice
  • Dulbecco's modified Eagle medium (DMEM; e.g., Invitrogen)
  • Tissue freezing medium (see recipe)
  • Isopropanol
  • 10× collagenase enzyme stock (see recipe)
  • 100× hyaluronidase enzyme stock (see recipe)
  • Digestion buffer (see recipe)
  • TAC solution (see recipe)
  • DMEM/F12 medium (unsupplemented; e.g., Invitrogen)
  • HBEC medium (see recipe)
  • HBEC freezing medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; Invitrogen, cat. no. 14040‐141)
  • 0.05% trypsin‐EDTA (Invitrogen, cat. no. 25300‐054)
  • Hyclone Hanks' balanced salt solution (HBSS; Thermo Scientific, cat. no. SH30268.02)
  • Characterized fetal bovine serum (FBS), heat inactivated (Thermo Scientific, cat. no. SH30071.03)
  • 10‐cm petri dishes, sterile
  • Disposable scalpels (no.10 blades)
  • Razor blades, sterile
  • 1‐ to 1.5‐ml cryovials, sterile
  • Nalgene Cryo 1°C freezing container filled with isopropanol (Nalgene, cat. no. 5100‐0001)
  • Balance for weighing tissue
  • Disposable cell lifter (Fisher, cat. no. 08‐100‐240), sterile
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • 37°C shaker/incubator
  • Centrifuge
  • 24‐well standard tissue culture plates
  • Light microscope
  • Additional reagents and equipment for trypan blue exclusion test of cell viability (Strober, )

Basic Protocol 2: Processing Human Body Fluid (e.g., Pleural Effusion, Ascites)

  Materials
  • Fresh human effusion samples (handle under BSL‐2 conditions)
  • TAC solution (see recipe)
  • Hyclone Hanks' balanced salt solution (HBSS; Thermo Scientific, cat. no. SH30268.02)
  • Dulbecco's phosphate‐buffered saline (DPBS; Invitrogen, cat. no. 14040‐141)
  • HBEC freezing medium (see recipe)
  • 50‐ml conical tubes, sterile
  • 500‐ml centrifuge bottles (Nalgene, cat. no. 3122‐0500)
  • Light microscope
  • 1‐ to 1.5‐ml cryovials, sterile
  • Additional reagents and equipment for freezing cells ( protocol 1)

Basic Protocol 3: Characterization of Single Cells Isolated from Human Tumors, Body Fluid, or from Tumor Graft Tissue Using Fluorescence Activated Cell Sorting (FACS)

  Materials
  • Solid tumor or body fluids
  • Modified M87 medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; Invitrogen, cat. no. 14040‐141)
  • 0.05% trypsin‐EDTA (Invitrogen, cat. no. 25300‐054)
  • Hyclone Hanks' balanced salt solution (HBSS; Thermo Scientific, cat. no. SH30268.02)
  • Characterized fetal bovine serum (FBS), heat‐inactivated (Thermo Scientific, cat. no. SH30071.03)
  • All of the reagents/antibodies listed in Table 14.23.3
  • Swinging‐bucket centrifuge
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 10‐cm tissue culture treated polystyrene cell culture dish (BD Falcon, cat. no. 353003)
  • 100‐µm cell strainer (BD Falcon, cat. no. 352360)
  • 12 × 75–mm round‐bottom polystyrene FACS tube (BD Falcon, cat. no. 352008)
  • 4‐color FACS machine capable of detecting FITC, PE, 7‐AAD, and APC fluorochromes (also see Coligan et al., , Chapter 5)
  • Additional reagents and equipment for trypan blue exclusion test of cell viability (Strober, ) and flow cytometry (Coligan et al., , Chapter 5)

Basic Protocol 4: Subcutaneous Implantation of Estrogen Pellets in Mice Receiving Estrogen Receptor–Positive Tumors

  Materials
  • Clidox−S disinfectant (Pharmacal Research Laboratories, http://www.pharmacal.com/Clidox.htm)
  • 3‐ to 4‐week‐old NOD/SCID female mice (The Jackson Laboratory, stock #1303)
  • Isoflurane (Baxter, cat. no. 1001936040)
  • Depilatory cream (such as Nair)
  • 70% ethanol
  • Povidone‐iodine (betadine) swabsticks (widely available)
  • Estrogen pellets, stored on ice (see protocol 12 for preparation of pellets)
  • Lidocaine‐bupivacaine mixture (see recipe)
  • Dissecting scissors, sterile
  • Forceps, sterile
  • Hot bead sterilizer (Fine Science Tools, cat. no. 18000‐45)
  • Sterile 7‐mm wound clips (Fine Science Tools, cat. no. 12031‐07)
  • Wound clip applicator (Fine Science Tools, cat. no. 12032‐07)
  • Wound clip remover (Fine Science Tools, cat. no. 12033‐00)
  • Heating pad (water circulating)
  • Surgical platform, such as a foam board
  • Anesthetic vaporizer (VetEquip, cat. no. 911103)
  • Compressed oxygen
  • Anesthesia induction chamber (VetEquip, cat. no. 941444)
  • Label tape
  • Cotton swabs, sterile

Basic Protocol 5: Implantation of Breast Tumor Tissue Fragments into Mice

  Materials
  • Tumor samples for implantation fresh or frozen (see Basic Protocols protocol 11 and protocol 22)
  • NOD/SCID mice (The Jackson Laboratory, stock #1303); alternatively, the more immune‐compromised NOD/SCID/IL2Rγ‐/‐ (NSG) mice (The Jackson Laboratory, stock #5557) can be used
  • HBEC medium (see recipe)
  • 70% ethanol
  • Clidox−S disinfectant (Pharmacal Research Laboratories, http://www.pharmacal.com/Clidox.htm)
  • Isoflurane (Baxter, cat. no. 1001936040)
  • Depilatory cream (such as Nair)
  • Povidone‐iodine (betadine) swabsticks (widely available)
  • Lidocaine‐bupivacaine mixture (see recipe)
  • Sterile sample tubes, e.g., 1.5‐ml microcentrifuge tubes
  • 50‐ml conical tubes, sterile
  • Heating pad (water circulating)
  • Dissecting scissors, sterile
  • Forceps, sterile
  • Sterile 7‐mm wound clips (cat. no. 12031‐07), wound clip applicator (cat. no. 12032‐07), and would clip remover (cat. no. 12033‐00; all from Fine Science Tools)
  • Hot bead sterilizer (Fine Science Tools, cat. no. 18000‐45)
  • Surgical platform, such as a foam board
  • Anesthetic vaporizer (VetEquip, cat. no. 911103)
  • Anesthesia induction chamber (VetEquip, cat. no. 941444)
  • 9‐mm nose cone (VetEquip, cat. no. 921609)
  • Label tape
  • Cotton swabs, sterile
  • Terumo U‐100 insulin syringes with 27‐G × 1/ 2 in. needles, sterile
  • Battery‐operated small‐vessel cauterizer (Fine Science Tools, cat. no. 18000‐00)
  • Calipers for measurement of tumor size
  • Additional reagents and equipment for preparing tumor tissue ( protocol 1)

Basic Protocol 6: Transplanting Tumor Cells and Organoids into Mice

  Materials
  • Organoids or cells from protocol 1 or 2
  • Growth factor–reduced Matrigel (BD, cat. no. 354230; or see protocol 12 for preparation of EHS matrix)
  • NOD/SCID mice (The Jackson Laboratory, stock #1303); alternatively, the more immunocompromised NOD/SCID/IL2Rγ‐/‐ (NSG) mice (The Jackson Laboratory, stock #5557) can be used
  • Terumo U‐100 insulin syringes with 27‐G × 1/ 2 in. needles, sterile
  • Additional reagents and equipment for implantation of breast tumor fragments into mice (Basic Protocol protocol 5)

Basic Protocol 7: Harvesting Tumorgrafts from Mice

  Materials
  • 70% ethanol
  • Tumor‐bearing mouse (see protocols above)
  • 4% paraformaldehyde (PFA; appendix 3D)
  • Liquid nitrogen
  • Tissue freezing medium (see recipe)
  • Dissecting equipment
  • Cotton swabs
  • Calipers for measuring tumor
  • Balance for weighing tumor
  • Plastic petri dish (Fisher Scientific, cat. no. 08‐757‐14)
  • 15‐ and 50‐ml conical tubes, sterile
  • 1‐ to 1.5‐ml cryovials, sterile
  • Hyclone DMEM/F12 (1:1) with HEPES (Thermo Scientific, cat. no. SH30023.01)
  • Additional reagents and equipment for CO 2 asphyxiation of the mouse (Donovan and Brown, ) and freezing of samples ( protocol 1, “b” steps)

Basic Protocol 8: Producing and Utilization of Lentiviruses for Transduction of Tumor Cells

  Materials
  • HEK 293T cells (ATCC #CRL‐11268)
  • DMEM medium with high glucose and L‐glutamine (Invitrogen, cat. no. 11965‐092)
  • Characterized, heat‐inactivated fetal bovine serum (FBS) (Thermo Scientific, cat. no. SH30071.03)
  • Dulbecco's phosphate‐buffered saline (DPBS; Invitrogen, cat. no. 14040‐141)
  • 0.05% trypsin‐EDTA (Invitrogen, cat. no. 25300‐054)
  • Polyethylenimine (PEI), branched, average mol. wt. ∼25,000 by LS (Sigma, cat. no. 408727‐100 ml)
  • OPTI‐MEM reduced serum medium with L‐glutamine (Invitrogen, cat. no. 31985‐070)
  • 0.5 to 1 µg/µl pHIV‐Luc‐ZsGreen (Addgene, cat. no. 39196)
  • 0.5 to 1 µg/µl solution of pMDLg/pRRE 3rd‐generation packaging plasmid (Addgene, cat. no. 12251)
  • 0.5 to 1 µg/µl solution of pRSV‐Rev 3rd‐generation packaging plasmid (Addgene, cat. no. 12253)
  • 0.5 to 1 µg/µl solution of pCMV‐VSV‐G envelope plasmid (Addgene, cat. no. 8454)
  • Standard bleach
  • Hyclone DMEM/F12 medium with HEPES (Thermo Scientific, cat. no. SH30023.01)
  • 4% paraformaldehyde (PFA)
  • Hyclone Hank's Balanced Salt Solution (HBSS; Thermo Scientific, cat. no. SH30268.02)
  • DNase I (AMRESCO, cat. no. 0649, http://www.amresco‐inc.com/)
  • 10× polybrene (hexadimethrine bromide, Sigma, cat. no. H9268) solution: 10 mg/ml in DPBS, filter sterilized and stored in aliquots at −20°C
  • 10‐cm tissue culture treated polystyrene cell culture dish (BD Falcon, cat. no. 353003)
  • Hemacytometer
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge with swinging‐bucket rotor, refrigerated
  • 0.45‐µm cellulose acetate filter membrane and 150‐ml bottle (Corning, cat. no. 431155)
  • Ultra clear 38.5 ml, 25 × 89 mm thin wall ultracentrifuge tubes (Beckman Coulter, cat. no. 344058)
  • Balance
  • Ultracentrifuge (Beckman Coulter Optima LE80k with swinging‐bucket SW28 rotor)
  • 0.5‐ml microcentrifuge tubes
  • 6‐well tissue culture treated polystyrene cell culture plate (BD Falcon, cat. no. 353224)
  • 6‐well ultra low‐attachment plates (Corning, cat. no. 3471)
  • 100‐µm cell strainer (BD Falcon, cat. no. 352360)
  • 12 × 75–mm round‐bottom polystyrene FACS tube (BD Falcon, cat. no. 352008)
  • FACS machine (flow cytometer) capable of detecting ZsGreen (488 nm) fluorescence (also see Coligan et al., , Chapter 5)
  • Light microscope
  • Additional reagents and equipment for counting cells using a hemacytometer (Strober, ), flow cytometry (Coligan et al., , Chapter 5), and preparation of organoids ( protocol 1)

Basic Protocol 9: Three‐Dimensional Primary Tumor Cultures

  Materials
  • Vial of cryopreserved human primary tumor cells
  • Modified M87 medium (see recipe)
  • Organoids, dissociated tumor cells, or pleural effusion cells (see Basic Protocols protocol 11 and protocol 22)
  • Growth factor‐reduced Matrigel (see recipe)
  • Test compounds
  • Vehicle (e.g., DMSO)
  • Cell Titer 96 aqueous non‐radioactive cell proliferation assay (MTS Assay) (Promega, cat. no. G5421)
  • Live/dead viability/cytotoxicity kit for mammalian cells (Invitrogen, cat. no. L‐3224)
  • 15‐ and 50‐ml conical tubes, sterile (e.g., BD Falcon)
  • 24‐well ultra‐low‐cluster (adhesion) flat‐bottom plates (Corning Costar, cat. no. 3473)
  • Centrifuge
  • Light microscope
  • 55‐ml disposable reagent reservoir (Bioexpress, cat. no. B‐0812‐7)
  • 96‐well untreated clear U‐bottom plates (BD Falcon, cat. no. 351177)
  • 12‐channel pipettor
  • 50‐ml multichannel pipetting reservoir
  • 96‐well clear flat‐bottom plates (BD Falcon, cat. no. 353915)
  • 96‐well black Cell Star clear flat‐bottom plate (Greiner Bio‐One, cat. no. 655090)
  • Plate reader capable of reading absorbance at 490 nm
  • Confocal microscope
  • Additional reagents and equipment for counting cells using a hemacytometer (Strober, )

Support Protocol 1: Obtaining Fresh Breast Tumor (e.g., Surgical Specimens, Body Fluids, and Metastases) from Patients

  Materials
  • Dewar flask of liquid nitrogen
  • RNAlater RNA stabilization reagent (Applied Biosystems, cat. no. AM7021)
  • RPMI 1640 (Fisher Scientific, cat. no. SH30255FS)
  • Bleach
  • 70% ethanol
  • Collection kit: (all reagents and supplies must be sterile)
    • Latex gloves
    • Disposable bibs
    • Sterile gauze
    • Swabs
    • Long, straight trimming blades
    • Sterile scalpels
    • Tissue culture dishes
    • Sterile forceps
    • 50‐ml conical vials
    • Sterile blue, green, and black ink
  • RNase/DNase‐free cryovials
  • 50‐ml conical tubes (e.g., BD Falcon)

Support Protocol 2: Preparation of Estrogen Pellets

  Materials
  • Beeswax (Sigma, cat. no. 243221)
  • 70% ethanol
  • Estradiol (1,3,5{10}‐Estratriene‐3, 17 β‐diol, Sigma, cat. no. E8875)
  • 22‐ml glass sample vial
  • Hot plate/magnetic stirrer
  • Aluminum foil
  • Dry ice
  • Weigh boats
  • Magnetic stir bar, sterile
  • Glass Pasteur pipets
  • Bunsen burner

Support Protocol 3: Preparation of Growth Factor–Reduced EHS Matrix

  Materials
  • 10 g Englebreth‐Holm‐Swarm (EHS) sarcoma tumor tissue isolated from mice (ATCC #CRL‐2108)
  • 3.4 M NaCl buffer (see recipe)
  • 2 M urea buffer (see recipe)
  • Tris‐buffered saline (TBS; see recipe)
  • Ammonium sulfate powder (Sigma, cat. no. A4418)
  • Chloroform (VWR, cat. no. BDH1109)
  • 70% ethanol
  • 50‐ml centrifuge bottle (Nalgene, cat. no. 3119‐0050)
  • Electric tissue homogenizer (Fisher Scientific PowerGen 125)
  • Refrigerated centrifuge with Beckman JA‐25.50 rotor
  • 250‐ml centrifuge bottle (Nalgene, cat. no. 3120‐0250)
  • 1.5‐in. magnetic stir bar
  • Stir plate (Thermolyne Nuova II)
  • Dialysis tubing (8000 MWCO; Spectrum Labs, cat. no. 132114)
  • Snap closures for dialysis tubing
  • 1‐liter glass beakers
  • DMEM medium with high glucose and L‐glutamine (Invitrogen, cat. no. 11965‐092)
  • Scissors and hemostat, sterilized
  • 15‐ml conical tubes, sterile
NOTE: It is important that every step of the protocol be conducted on ice or at 4°C, or the solutions will solidify and the entire batch will be unusable.
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Literature Cited

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