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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 CO₂ 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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Figures

Figure 14.23.1 Tumorgrafts resemble the original tumors from which they were derived. A representative ER^‐ PR^‐ HER2^‐ tumor graft (HCI−001) is shown in comparison to the original patient sample. The tumor ID and the original clinical diagnosis for ER, PR, and HER2 are shown at the top. Sections from the patient's primary breast tumor (patient), and from representative tumor grafts from the same patient (graft). Stains shown are hematoxylin and eosin (H&E) as well as antibody stains for ER, PR, HER2, cytokeratin (CK), E‐cadherin (E‐cad), β‐catenin (β‐cat), and human‐specific vimentin (hVim). Positive antibody signals are brown in color, with hematoxylin (blue) counterstain. Some images are shown at higher magnification to visualize nuclear staining. All scale bars correspond to 100 µm. This figure was reprinted from DeRose et al. () with permission

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Figure 14.23.2 Example of breast tumor tissue isolated from a patient. (A ) Surgical sample containing tumor tissue (white‐pink color) and fat (yellow color). (B ) Fragments of the same tissue sample following removal of the fat and dissection into approximately 4 × 2‐mm pieces, ready for implantation into mammary fat pads. The halo surrounding the tissue is due to the fluid used to keep the samples moist during processing.

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Figure 14.23.3 Metastatic tumor cells collected from a pleural effusion and viewed using light microscopy. These breast tumor cells aggregated into organoid‐like structures, which were easily separated from single cells such as leukocytes that were present in the original specimen. Scale bar is 100 µm.

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Figure 14.23.4 (A ) PE1007070 and (B ) PE1008032, which are primary cells isolated from pleural effusions from two separate patients, were analyzed by FACS for 7‐AAD and lineage markers in combination with either CD44/CD24 or EPCAM/CD49f. (C ) Primary cells isolated from pleural effusions from seven patients were analyzed by FACS for lineage markers and CD44/CD24.

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Figure 14.23.5 Schematic showing location of mouse mammary fat pads, surgical incisions, and sites for tissue/cell transplants and the estrogen pellet implant. Mouse diagram was adapted from Hummel et al. () with permission of The Jackson Laboratory.

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Figure 14.23.6 Analysis of the viability of patient‐derived breast cancer organoids in 3‐D culture following drug treatment. Primary tumor organoids were embedded in Matrigel and grown in 3‐D culture. Organoids were treated with (A ) DMSO or valproic acid for 4 days, or (B ) DMSO or taxol for 7 days, and imaged using both DIC (bottom rows) and fluorescence microscopy (top rows). Live/dead discrimination was performed using calcein AM (green) to mark viable cells, and ethidium bromide homodimer‐1 (red) to identify dead cells. Scale bars represents 20 µm. Panel A was adapted from Cohen, et al. () with permission.

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