patient-derived models of human breast · 2020-05-21 · models of human breast cancer 14.23.4...

UNIT 14.23Patient-Derived Models of Human BreastCancer: Protocols for In Vitro and In VivoApplications in Tumor Biology andTranslational Medicine

Yoko S. DeRose,1,5 Keith M. Gligorich,2,5 Guoying Wang,1 AnnGeorgelas,3 Paulette Bowman,3 Samir J. Courdy,4 Alana L. Welm,1 and BryanE. Welm2

1Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, SaltLake City, Utah2Department of Surgery, Huntsman Cancer Institute, University of Utah, Salt Lake City,Utah3Tissue Resource and Applications Core Shared Resource Facility, Huntsman CancerInstitute, University of Utah, Salt Lake City, Utah4Research Informatics, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah5 These authors contributed equally to this work.

ABSTRACT

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

Keywords: breast cancer � tumorgraft � organoid � 3D culture � Matrigel �

EHS matrix � biospecimen � tissue repository � tissue collection � xenograft

INTRODUCTION

Model systems are essential for discovery, development, and testing of new therapiesfor breast cancer. Generating the appropriate models for particular applications is chal-lenging, as all models have both advantages and disadvantages. (Voskoglou-Nomikoset al., 2003; Gupta and Kuperwasser, 2004). Immortalized cell lines are used routinelyto study breast cancer, are easy to grow and manipulate, and are ideal for examining themolecular mechanisms of tumor cell biology (Burdall et al., 2003; Holliday and Speirs,2011). Likewise, implantation of breast cancer cell lines into mice for in vivo studies isa useful way to study mechanisms of tumor growth, progression, metastasis, and drugresponse (Clarke, 1996; Kerbel, 2003; Cespedes et al., 2006). A caveat in the use of celllines, however, is their prolonged adaptation to tissue culture conditions that has led togenetic and epigenetic alterations over time (Neve et al., 2006; Kao et al., 2009; Tsujiet al., 2010). It is also necessary to grow xenografts in immune-compromised mice invivo where the tumor environment is different from that of native tumors (Clarke, 1996;Kerbel, 2003; Cespedes et al., 2006).

Current Protocols in Pharmacology 14.23.1-14.23.43, March 2013Published online March 2013 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471141755.ph1423s60Copyright C© 2013 John Wiley & Sons, Inc.

Cellular andAnimal Modelsin Oncology andTumor Biology

14.23.1

Supplement 60

Models of HumanBreast Cancer

14.23.2

Supplement 60 Current Protocols in Pharmacology

H&EH&E

H&EH&E

ERER

HCI-001 (ER , PR , HER2 )

Pat

ient

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PRPR

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HER2HER2

HER2HER2

CKCK E-cadE-cad -cal-cal hVimhVim

CKCK E-cadE-cad -cal-cal hVimhVim

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ER

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PR

HER2

HER2

CK E-cad -cal hVim

CK E-cad -cal hVim

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ftP

atie

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raft

Figure 14.23.1 Tumorgrafts resemble the original tumors from which they were derived. A rep-resentative ER-PR-HER2- tumor graft (HCI−001) is shown in comparison to the original patientsample. The tumor ID and the original clinical diagnosis for ER, PR, and HER2 are shown atthe top. Sections from the patient’s primary breast tumor (patient), and from representative tumorgrafts from the same patient (graft). Stains shown are hematoxylin and eosin (H&E) as well asantibody stains for ER, PR, HER2, cytokeratin (CK), E-cadherin (E-cad), β-catenin (β-cat), andhuman-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. (2011) withpermission.

Genetically engineered mouse models, on the other hand, allow the study of tumordevelopment and progression in immune-competent mice (Borowsky, 2011; Caligiuriet al., 2012). However, most mouse genetic models rely on the expression of a single,potent oncogene to drive the tumor, and frequently do not recapitulate the full phenotypicrepertoire of the human tumor or metastatic patterns seen in breast cancer patients. Areview of various cancer models and their utility for drug development can be found inUNIT 14.22, and also as a summary in Table 14.23.1.

We have recently reported that directly implanting breast tumor tissues derived frompatients into mouse mammary fat pads results in a remarkable recapitulation of humanbreast cancer. These “tumorgrafts” closely resemble the original tumors from the patientby: (i) maintaining their clinical markers and histopathologies; (ii) maintaining hormonedependence or independence; (iii) maintaining their gene expression and DNA copynumber variations; and (iv) recapitulating clinically relevant metastasis (DeRose et al.,2011 and Fig. 14.23.1). Although the techniques to generate these tumorgrafts can bechallenging, the effort is worthwhile as they are the best currently available model ofhuman breast cancer. Such a resource is likely to become a valuable asset for drugdiscovery efforts, particularly for preclinical animal studies to assess the effectivenessof lead compounds on specific cancer subtypes. In this way, tumorgrafts could helpdistinguish cancer subtypes that are either responsive or unresponsive to a compound,


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Current Protocols in Pharmacology Supplement 60

Table 14.23.1 Comparison of Different Breast Cancer Models

Breast cancer model Strengths Weaknesses

Carcinogen-induced mousemodel

Immune-competent mouse anddiverse tumor subtypes are generated

Tumor is not human or may not reflect thegenetic variations that occur in humans; shorttumor evolution may not replicate events thatoccur during the longer evolution of humantumors; intra-tumor heterogeneity is oftenlimited; requires genetic tools; laborious togenerate; expensive; and limited number ofcancer types are modeled

Genetically engineeredmouse models (GEMM)

Defined oncogenic driver; limitedvariability between tumors; induciblemodeling possible; targetedoncogenesis in specific cell types;immune-competent mouse; ability tomodel early events in oncogenesis

Tumor is not human or may not reflect thegenetic variations that occur in humans; shorttumor evolution may not replicate events thatoccur during the longer evolution of humantumors; laborious to generate; expensive;limited number of cancer types currentlymodeled

Human cell line xenograft Human tissue; cell of origin andoncogenic drivers are directlyassociated with human cancer; cellsare easily grown and manipulated inculture; many cells lines wellcharacterized in the literature, and canbe genetically modified and selected

Genetic drift; selective pressure from cellculture; requires immune-compromised mice

Patient-derived tumorgrafts Human tissue; cell of origin andoncogenic drivers directly associatedwith human disease; can begenetically modified; naturalevolution in patients; bothchemo-naı̈ve and standard-of-carechemo-resistant tissue; no selectivepressure from in vitro culturing;molecular and genetic markers highlyrepresentative of primary tumors;potential for personalized medicine

Not all subtypes grow well in mice; requiresimmune-compromised mice; laborious;expensive; establishment of bio-bank requiressignificant coordination between physiciansand departments, quality control of specimenprocessing, IBC regulatory approval for tissuecollection, HIPPA, PHI; intellectual propertyissues

and enable clinical trials to be designed around the most appropriate patient population.While a significant investment must be made to establish a bank of transplantable andsubtype-diverse tumorgrafts, better prediction of a drug’s effectiveness should lead tomore successful clinical trials, which would appreciably outweigh the initial investment.Still, the validity and practicality of primary tumorgrafts remains to be verified in a drugdevelopment program.

Tumors removed from patients can be processed in many different ways (Table 14.23.2).Thus, it is important to identify the most appropriate processing technique for a re-search purpose. Here, we describe three types of tissue processing: tumor fragments,organoids, and single-cell suspensions. The processing occurs sequentially. Tumors arefirst removed from patients and manually dissected into tissue fragments that can beused for tissue transplantation. Processing of tumor fragments by mincing and enzy-matic digestion with collagenase and hyaluronidase leads to tumor organoids, which canbe used for transplantation or 3-D culturing. Finally, tumor organoids can be digestedwith trypsin/EDTA to generate a single-cell suspension for use in transplantation, cell


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Table 14.23.2 Processing Methods for Patient-Derived Primary Tumor Tissue

Tumor fractionProcessingtechnique

Processing method Composition of fraction Usage

Fragment Manual Tumor is manually cutinto fragments withscalpel or razor

>1 mm-diameter tumor fragments;cell contacts are maintained; maycontain blood vessels, stroma,immune cells, extracellular matrix

Transplantation

Organoid Manual andenzymatic

Tumor fragments arechopped and thendigested withcollagenase andhyaluronidase

50- to 200-μm tumor fragment; cellcontacts are maintained; somestroma may be present

Transplantationand/or 3-D culturing

Single cells Manual andenzymatic

Organoids are isolatedand then digested withtrypsin/EDTA

Isolated tumor cells; cell contactsare lost; diverse tumor and stromalcell populations present butdispersed in suspension

Transplantation; 2-Dculturing; viralinfection; FACS

culturing, virus infection, and FACS. Thus, tumor tissue is serially processed throughstages as needed for specific experimental applications.

Outlined in the present unit are procedures for processing human breast cancer tis-sue and implanting it into mice (Basic Protocol 1); processing human body fluid (e.g.,pleural effusions) for transplantation or storage (Basic Protocol 2); characterizing singlecells isolated from human breast tissue or pleural effusions by cell surface markers (BasicProtocol 3); implanting estrogen pellets subcutaneously into mice (Basic Protocol 4); im-planting breast tissue into cleared mammary fat pads (Basic Protocol 5); and, implantingbreast cancer cells with Matrigel into cleared mammary fat pads (Basic Protocol 6). De-tails are also provided for the isolation of tumor cells from tumor grafts using enzymaticdigestion (Basic Protocol 7); producing and transducing the cells with lentiviruses (BasicProtocol 8); and growing tumorgraft or primary patient organoids in three-dimensional(3-D) cultures for drug discovery and validation (Basic Protocol 9). Support protocolsinclude describing the isolation and storage of human breast tumor tissue (Support Pro-tocol 1); preparation of estrogen pellets (Support Protocol 2); and preparation of growthfactor-reduced Engelbreth-Holm-Swarm (EHS) tumor matrix (Support Protocol 3).

NOTE: All human patient tissue samples must be collected from informed, consentingpatients under an approved Institutional Review Board for Human Participants (IRB)protocol. Tissue collection at the Huntsman Cancer Institute is coordinated by the TissueResource and Applications Core shared resource facility using protocols described inSupport Protocol 1. Specific training is provided to all staff members involved in obtainingpatient consent, and in the collection and processing of tissue to ensure the acquisitionof high-quality biospecimens and compliance with the Health Insurance Portability andAccountability Act (HIPAA) and the protection of Personal Health Information.

NOTE: All protocols using live animals must first be reviewed and approved by an Insti-tutional Animal Care and Use Committee (IACUC) and must follow officially approvedprocedures for the care and use of laboratory animals (also see APPENDIX 4).

NOTE: All solutions and equipment that come into contact with cells must be sterile, andproper aseptic technique used for work with cell cultures.

NOTE: All culture incubations should be performed in a humidified 37◦C, 5% CO2

incubator unless otherwise specified.


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BASICPROTOCOL 1

PROCESSING HUMAN BREAST TUMORS AND TUMORGRAFTS INTOFRAGMENTS, ORGANOIDS, AND SINGLE CELLS

It is typically preferable to store primary patient and xenograft tumor tissue as fragmentssince they have a higher transplantation success rate than either organoids or single-cellsuspensions (DeRose et al., 2011, and authors’ unpub. observ.). All fresh tumor tissuesamples must be kept on ice and processed in sterile conditions on the day of collection.All tissue processing is performed in a biosafety cabinet (hood) using Biosafety Level2 (BSL2) techniques. See Support Protocol 1 for information on the collection andcataloging of primary human tissue.

Materials

Human breast primary tumor tissue, metastasis tissue, effusion samples (fresh; thequantity usually varies) or fresh tumorgrafts derived from mice

Dulbecco’s modified Eagle medium (DMEM; e.g., Invitrogen)Tissue freezing medium (see recipe)Isopropanol10× 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, sterileDisposable scalpels (no.10 blades)Razor blades, sterile1- to 1.5-ml cryovials, sterileNalgene Cryo 1◦C freezing container filled with isopropanol (Nalgene, cat. no.

5100-0001)Balance for weighing tissueDisposable cell lifter (Fisher, cat. no. 08-100-240), sterile50-ml conical polypropylene centrifuge tubes (e.g., BD Falcon)37◦C shaker/incubatorCentrifuge24-well standard tissue culture platesLight microscope

Additional reagents and equipment for trypan blue exclusion test of cell viability(Strober, 1997)

To process solid tissue into fragments

1a. Place the solid tumor tissue (size can vary) in a 10-cm petri dish, pouring sterileserum-free DMEM medium on the tissue to keep it moist.

2a. Cut the tumor into pieces using a scalpel or razor blade, removing necrotic tissue ifpresent.

Necrotic human tissue is typically whiter and softer compared to the surrounding tumortissue since it is less vascularized. Necrotic tumorgraft tissue from mice is normally darkerand softer compared to the surrounding tumor.


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A B

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

3a. Proceed to transplantation (Basic Protocol 5) or cryopreservation (step 4a, below), asneeded.

4a. For cryopreservation: Cut the tumor tissue into 4 × 2–mm fragments (Fig. 14.23.2).Place five fragments in a cryovial and fill with tissue freezing medium (do not confusewith HBEC freezing medium). Freeze the cells by slowly decreasing the temperature1◦C/min (this can be done by placing the vials in a Nalgene Cryo 1◦C cell-freezingcontainer filled with room temperature isopropanol). Place the container at −80◦Covernight before transferring the vials to liquid nitrogen cryotanks.

We freeze five fragments per vial so that each vial is sufficient for implantation into fivemice when thawed. Do not refreeze tissue fragments.

To process solid tissue into organoids

1b. Weigh the tissue.

The quantity of tissue used will depend on the amount obtained from surgery and theamount reserved as fragments.

2b. Mince the tissue using a criss-cross motion with two disposable scalpels until finelychopped, then transfer the minced tissue to a 50-ml conical centrifuge tube using acell lifter.

3b. Add digestion reagent(s) (collagenase and/or hyaluronidase solution) to a finalvolume of 10 ml per g of tissue.

i. For primary human specimens: Add 10× collagenase solution and 100×hyaluronidase solution to a final concentration of 1× each.

ii. For tumor graft tissue: Add only 10× collagenase solution to a final concentrationof 1×.


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4b. Close the tube with a cap and wrap the cap with Parafilm (Parafilm helps to preventleakage and potential bacteria/yeast contamination of the area around the cap). Placethe tube on its side in a 37◦C shaker at low to moderate speed (e.g., 200 rpm).

5b. Determine the incubation time, which varies between primary human tissue andtumorgraft tissue.

i. For primary human samples from patients: Due to the extensive stromal component,human tumor samples require more time in the digestion reagents. Shake/incubatehuman primary tissue overnight (∼18 hr) in the digestion reagents.

ii. For tumorgraft tissue: Tumorgrafts (derived from mice) contain less stroma and aredigested more easily than primary human samples. Shake for 40 min and determineif tissue is well digested. If not, incubate longer, checking every 5 to 10 min.

Monitor the digestion by gross observation and light microscopy of the digestion reac-tions. Grossly large, undigested material can sometimes be seen in the digestion reactions.The large undigested tissue may contain cell debris, DNA from lysed cells, fat, and ex-tracellular matrix. This material typically forms large strands rather than chunks, andwill not digest or produce many organoids. Organoids are best visualized by microscopy.Take a 25-μl sample of the digestion medium and dilute 1:10 to 1:100 in DPBS, andplace 25 μl of the dilution in an empty well of a 24-well standard tissue culture dish. Lookfor large refractory clusters of epithelial cells, called organoids. More digestion may beappropriate if most of the organoids are >200 μm in diameter. If the tissue does notdigest well, remove all undigested fragments and debris by straining the digested tissueinto a new tube using a 100-μm cell strainer. Retain the digestion reaction that passesthrough the strainer and visualize under a microscope as described above. If organoidsare present, continue the protocol with this suspension at step 6b. Place the strainedmaterial in a 50-ml conical tube, add fresh digestion buffer, then shake for 1 to 3 morehours to recover more digested tissue. Continue with step 6b when most organoids are<200 μm diameter.

6b. Centrifuge 5 min at 530 × g, room temperature, to pellet the cells.

7b. Aspirate the supernatant, which contains fat. If the pellet contains red blood cells(observed as a red layer on top of the pellet), resuspend the pellet in 5 to 10 ml ofTAC solution and incubate 3 to 10 min in a 37◦C water bath. Centrifuge 5 min at530 × g, room temperature. Repeat this step until the red blood cells are no longervisible.

Steps 8b to 9b in this protocol function to separate organoids, which contain most ofthe tumor epithelium, from stromal cells and debris using a process called differentialcentrifugation. The two key principles are: (1) the collagenase/hyaluronidase digestionpreferentially disrupts cell contacts between stromal cells, causing their release as singlecells, while tumor epithelium remains mostly adherent, resulting in large (100- to 200-μmdiameter) clusters of tumor cells (organoids); and (2) the difference in weight betweensingle cells/debris and organoids enables fractionation by centrifugation. This processmust be optimized with different centrifuges, since acceleration and deceleration kineticsvary with each instrument.

8b. Resuspend the pellet in 10 ml of DMEM/F12 medium (with no supplements) andcentrifuge 10 to 30 sec at 530 × g, room temperature. Collect both the supernatant(suspension fraction) and pellet (organoid fraction). Collect the cells in the suspen-sion fraction by centrifuging 5 min at 530 × g, room temperature, and removing thesupernatant. Resuspend each fraction in 1 ml DMEM/F12 medium (with no supple-ments). Take a 10- to 25-μl sample of the resuspended fraction and dilute 1:10 to1:100 in DPBS. Place 25 μl of each dilution in an empty well of a 24-well standardtissue culture dish. Examine the quality of the fraction under a light microscope.

The organoid fraction should be more enriched for refractile, solid clusters of tumor cells,termed “organoids,” and there should be few single cells (Fig. 14.23.3). The suspension


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Figure 14.23.3 Metastatic tumor cells collected from a pleural effusion and viewed using lightmicroscopy. These breast tumor cells aggregated into organoid-like structures, which were easilyseparated from single cells such as leukocytes that were present in the original specimen. Scalebar is 100 μm.

fraction should be enriched in single cells and be relatively free of organoids. Continuefractionating by differential centrifugation as described in step 9b.

9b. Resuspend the pellet from the organoid fraction in 9 ml of DMEM/F12 medium (withno supplements) and centrifuge 10 to 30 sec at 530 × g, room temperature, to pelletthe cells. Centrifuge the suspension fraction 5 min at 530 × g, room temperature,to pellet the suspended cells. Analyze both samples as described in step 8b. Tofurther enrich organoids from single cells, perform differential centrifugation byrepeating this step three to four times until the desired level of organoid enrichmentis achieved.

The fractionation procedure is significantly impacted by the type/brand of centrifugeused due to differences in time-to-full-speed and time-to-stop. Thus, this step requiresoptimization of the centrifugation times for different centrifuges. The investigator mustalso determine a desired level of organoid enrichment for each experiment. For example,more highly enriched organoids have fewer stromal and immune cells in the preparation.Thus, it is beneficial to perform more differential centrifugations for applications suchas culturing organoids in 3-D, processing organoids into single cells for FACS analysis,and viral transduction. In contrast, it is not necessary to have highly enriched organoidsfor transplantation.

10b. Estimate the number of organoids in the final sample and calculate the approximatetotal number of organoids recovered from the tissue. Culture the 24-well platecontaining the 25-μl samples used to assess the fractionation procedure (step 8b)overnight in 0.5 ml/well of HBEC medium to determine whether the sample iscontaminated with bacteria or fungus.

Contamination can be a problem because samples are not handled using sterile tech-nique in most gross pathology labs. At our institution, we eliminated the problem ofcontamination by providing the lab with a sterile breast sample collection kit to be usedfor collection of viable specimens (see Support Protocol 1).


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11b. Proceed to cryopreservation (step 12b), transplantation (Basic Protocol 6), single cellprocessing (“c” steps, below), or 3-D cell culture (Basic Protocol 9), if applicable.

12b. For cryopreservation, resuspend the organoids in HBEC freezing medium in aliquotsof 1.5 ml containing up to 10,000 organoids per vial. Freeze the cells by slowlydecreasing the temperature 1◦C/min (this can be done by placing the vials in aNalgene Cryo 1◦C cell-freezing container filled with room temperature isopropanol).Place the container at −80◦C overnight before transferring the vials to liquid nitrogencryotanks. If there is a problem with the overnight culture (e.g., contamination withfungus or bacteria), discard the frozen organoids.

To process organoids into single cells

1c. Centrifuge the suspension containing organoids for 5 min at 400 × g, 4◦C.

2c. Aspirate the supernatant and carefully resuspend the pellet with 10 ml of DPBS bypipetting up and down on ice.

3c. Centrifuge the cells for 5 min at 400 × g at 4◦C.

4c. Aspirate the supernatant and carefully resuspend the pellet with 1 to 5 ml of 0.05%trypsin-EDTA. Place the conical tube in a 37◦C shaker/incubator at 150 to 300rpm. Every 5 min, remove a 10-μl aliquot of the suspension and observe under amicroscope. Continue the incubation if organoids are still present.

Some tumor organoids can take >20 min to dissociate into single cells and may requireaddition of more trypsin. However, longer incubation times with trypsin will reduce cellviability. To increase viability when long trypsin incubations are required, it may be nec-essary to separate single cells from clusters every 10 min (using differential centrifugationor cell straining). Single cells could be washed (as in step 5c) and stored on ice while theclusters are digested for a longer.

5c. Once single cells are obtained, add 10 ml of HBSS containing 2% FBS and centrifugethe cells 5 min at 400 × g, 4◦C.

6c. Aspirate the supernatant and carefully resuspend the pellet with 5 ml of HBSScontaining 2% FBS by pipetting up and down. Perform a trypan blue exclusion testusing a hemacytometer to determine the number of viable cells per ml (Strober, 1997).

7c. For cryopreservation, resuspend the pellet in HBEC freezing medium (do not confusewith tissue freezing medium) at a concentration of ∼5–10 × 106 cells per ml andfreeze in 1-ml aliquots as described above.

8c. Proceed to transplantation (Basic Protocol 6), FACS (Basic Protocol 3), lentiviralinfection (Basic Protocol 8), or 3-D culture (Basic Protocol 9).

BASICPROTOCOL 2

PROCESSING HUMAN BODY FLUID (e.g., PLEURAL EFFUSION, ASCITES)

In addition to solid tumors, patients with metastatic breast cancer can present with apleural effusion, which is a buildup of fluid and tumor cells in the pleural cavity. It is notunusual for 500 ml to 1.5 liters of effusion fluid to be collected from a single patient,which can contain large amounts of tumor cells. This fluid can be a very rich sourceof metastatic breast cancer cells, but can contain other contaminating cell types, suchas lymphocytes, macrophages, and fibroblasts. Therefore, cells isolated from pleuraleffusions or ascites fluid should be characterized by cell surface markers, as described inBasic Protocol 3.


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

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, sterile500-ml centrifuge bottles (Nalgene, cat. no. 3122-0500)Light microscope1- to 1.5-ml cryovials, sterile

Additional reagents and equipment for freezing cells (Basic Protocol 1)

1. Transfer the freshly acquired effusion fluid to a 50-ml conical tube on ice. For large-volume samples, use sterile 500-ml Nalgene centrifuge bottles.

2. Centrifuge the fluid for 5 min at 530 × g, 4◦C, and collect the pellet.

If using a rotor that is also used to centrifuge bacterial medium, be sure to disinfect theoutside of the bottles thoroughly with ethanol prior to performing the next steps, to preventcontamination.


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3. If the pellet contains red blood cells (observed in the top layer of the cell pellet),resuspend the pellet in 5 to 10 ml of TAC solution and incubate the sample for 3 to10 min in a 37◦C water bath. Centrifuge 5 min at 530 × g, room temperature. Repeatthis step until the blood cells are no longer visible.

4. Resuspend the pellet in 5 to 10 ml of HBSS or DPBS.

5. Centrifuge 5 min at 530 × g, room temperature.

6. Resuspend the pellet in 5 to 10 ml HBSS or DPBS and assess its content by plating25 μl into a dish and examining the cellular organization under a light microscope(organoids and single cells; Table 14.23.2 and Fig. 14.23.3).

The organization of tumor cells in pleural effusions/ascites varies significantly, rangingfrom organoid-like aggregates to fully dispersed single cells. In addition, the number oftumor and immune cells is highly variable (Fig. 14.23.4). Both the cellular organization(based on microscopy) and cell content (based on FACS, Basic Protocol 3) should berecorded for each effusion. Single cells can be prepared from organoids as described inBasic Protocol 1.

7. Centrifuge 5 min at 530 × g, room temperature.

8. Proceed to transplantation (Basic Protocol 6), FACS (Basic Protocol 3), lentiviralinfection (Basic Protocol 8), or 3-D culture (Basic Protocol 9).

9. For cryopreservation, resuspend the pellet in HBEC freezing medium (do not con-fuse with tissue freezing medium) to a concentration of approximately 1,000,000organoids or 5 to 10 million cells per ml and freeze in 1-ml aliquots as described inBasic Protocol 1.

BASICPROTOCOL 3

CHARACTERIZATION OF SINGLE CELLS ISOLATED FROM HUMANTUMORS, BODY FLUID, OR FROM TUMOR GRAFT TISSUE USINGFLUORESCENCE ACTIVATED CELL SORTING (FACS)

Primary human tumors typically contain heterogeneous cell populations, including stro-mal and immune cells, in addition to tumor cells with diverse tumorigenic capacity. Ithas been shown that these different populations can be characterized using cell-surfacemarkers such as epithelial cell adhesion molecule (EPCAM) and CD49f (α6 integrin),which can differentiate luminal (EPCAMHi/CD49fLow) versus basal/myoepithelial cells(EPCAMlow/CD49fHi) in normal breast tissue, and exhibit distinct expression in breastcancer subtypes (Keller et al., 2010). In addition, the cell-surface markers CD44Hi andCD24Low have been used to identify a population of breast cancer cells with enhancedtumor-initiating capabilities in mouse transplantation assays (Al-Hajj et al., 2003). Sinceevery patient sample may contain different populations of tumor cells as well as con-taminating nonepithelial cells, such as lymphocytes, macrophages, and fibroblasts, it isimportant to characterize single cells isolated from human breast tumors (Basic Protocol1), human body fluids (Basic Protocol 2), or tumorgraft tissue (Basic Protocol 8).

The protocol involves staining single-cell suspensions derived from tumors with a varietyof antibodies that differentially mark specific cell populations. For example, 7-AAD isused to exclude dead cells, and a cocktail of antibodies (CD2, CD3, CD10, CD16, CD18,CD31, CD64, and CD140b) is used to exclude nonepithelial cells (lineage positive),such as lymphocytes, macrophages, and fibroblasts. Simultaneously, the cells are stainedwith combinations of antibodies that recognize tumor markers, such as EPCAM, CD49f,CD44, and CD24, which are expressed on diverse tumor cell populations. The cellsuspension is then analyzed or sorted into enriched cell fractions by flow cytometry.An example of a typical FACS analysis of primary cells isolated from pleural effusionsfrom two patients is illustrated in Figure 14.23.4A and B. Additionally, the percentage


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Table 14.23.3 Antibody and Reagent Concentrations for FACS

Reagent Source Catalog no. Concentration

PE mouse anti-human CD2 BD Biosciences 555327 1:100 v/va







PE mouse anti-human CD140b BD Biosciences 558821 1:100 v/va

FITC mouse anti-human CD24 BD Biosciences 555427 1:50 v/v

APC mouse anti-human CD44 BD Biosciences 559942 1:50 v/v

FITC rat anti-human CD49f BD Biosciences 555735 1:50 v/v

APC mouse anti-human EPCAM BD Biosciences 347200 1:100 v/v

7-AAD staining solution BD Biosciences 559925 0.25 μg/106 cellsaAll of the PE conjugated antibodies are mixed together to generate the linage cocktail.

of lineage-positive (nonepithelial) and CD44Hi/CD24Low (tumor-initiating) cells fromseven different primary patients’ samples is presented in Figure 14.23.4C. These datademonstrate that every patient sample contains different percentages of lineage-positiveand CD44Hi/CD24Low populations. Typically, we do not utilize primary patient cells thatcontain high levels (>30% to 40%) of lineage-positive cells for the three-dimensionalprimary tumor cultures described in Basic Protocol 9.

Materials

Solid tumor or body fluidsModified 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 centrifuge15- 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., 2012, Chapter 5)

Additional reagents and equipment for trypan blue exclusion test of cell viability(Strober, 1997) and flow cytometry (Coligan et al., 2012, Chapter 5)

Culture freshly prepared tumor cells overnightThe cells are cultured overnight in order to allow them to recover from the tissue process-ing procedure (Basic Protocols 1 and 8) before performing FACS staining. Alternatively,


14.23.13


cells can be used directly after processing, but they may be more susceptible to dyingduring the staining procedure

1a. For freshly isolated tumor cells: Prepare fresh single tumor cells from solid tumor orbody fluids as described in Basic Protocol 1 or 2, then continue this protocol at step 5.

1b. For cryopreserved cells: Defrost a vial of cryopreserved human primary tumor cellsby submerging it in a 37◦C water bath for 2 to 3 min. As soon as the vial is thawed,transfer the contents to a 15-ml conical tube containing 10 ml of modified M87medium that has been prewarmed to 37◦C.

2. Centrifuge the cells for 5 min at 400 × g, room temperature.

3. Aspirate the supernatant and carefully resuspend the pellet into 1 ml of modified M87medium that has been prewarmed to 37◦C by pipetting up and down.

4. Perform a trypan blue exclusion test using a hemacytometer to determine the numberof viable cells per ml (Strober, 1997).

5. Adjust the concentration of cells to 4–6 × 106 cells in 10 ml by addition of modifiedM87 medium that has been prewarmed to 37◦C.

6. Add 10 ml of the cell suspension to a 10-cm tissue culture–treated dish. Incubate thecells at 37◦C in a 5% CO2 humidified incubator, overnight.

Collect cells after overnight incubation7. Remove the plate from the incubator and collect the medium, which may contain

nonadherent tumor cells, and transfer to a 50-ml conical tube on ice.

Some pleural effusions contain nonadherent tumor cells that do not need to be trypsinizedprior to FACS. However, if the nonadherent tumor cells are aggregated in clusters ororganoids, they should be processed into single cells as described in Basic Protocol 1,steps 1c to 8c. The presence or absence of adherent cells does not necessarily correlatewith the percentage of lineage positive (nonepithelial) cells.

8. To remove adherent cells, immediately wash the plate with 10 ml of DPBS andtransfer the solution to the 50-ml conical tube containing the cell suspension on ice.Add 1 ml of 0.05% trypsin-EDTA to the plate.

9. Incubate the cells with trypsin for 1 to 5 min at 37◦C until the adherent cells detach.Gently pipet up and down to mix the trypsin solution and transfer it to the 50-mlconical tube containing the suspension cells.

10. Wash the plate with 10 ml of HBSS containing 2% FBS and transfer the solution tothe 50-ml conical tube containing the cell suspension on ice.

11. Centrifuge the cells for 5 min at 400 × g at 4◦C.

12. Aspirate the supernatant and carefully resuspend the pellet with 5 ml of HBSScontaining 2% FBS by pipetting up and down. Perform a trypan blue exclusion testusing a hemacytometer to determine the number of viable cells per ml (Strober, 1997).

13. Adjust the concentration to 1.0 × 106 cells/ml by adding HBSS containing 2% FBSon ice and proceed to the staining, step 14.

If large adherent clusters of cells are present, they will need to be further processed intosingle cells. Centrifuge the cell suspension for 5 min at 400 × g, 4◦C, and resuspend thepellet in 1 ml of 0.05% trypsin-EDTA. Repeat steps 9 to 13 until the cells are disaggregated.DNA released from cell lysis during the trypsinization step may cause the medium tobecome viscous and difficult to handle, and may cause cells to clump together. If thisoccurs, add DNase I (10 μg/ml final concentration DNase I) to the medium and incubatefor ∼5 min at room temperature, then repeat steps 12 to 14.


14.23.14


Prepare cells for FACS analysis of CD44/CD24 and EPCAM/CD49f markers14. Add 1 ml of the single-cell suspension at 1.0 × 106 cells/ml to nine 15-ml conical

tubes (1.0 × 106 cells/sample) for each staining condition listed below. For theremainder of the staining procedure, keep the cells on ice and in the dark. Also, keepthe HBSS containing 2% FBS on ice.

a. No antibody

b. CD24-FITC (FL1 setup)

c. Lineage-PE Cocktail, which is a mixture of PE conjugated CD3, CD10, CD16,CD18, CD31, CD64, and CD140b antibodies (FL2 setup)

d. 7-AAD (FL3 setup)

e. CD44-APC (FL4 setup)

f. CD44/CD24 Analysis (7-AAD, Lineage PE cocktail, CD24-FITC and CD44-APC)

g. CD49f-FITC (FL1 setup)

h. EPCAM-APC (FL4 setup)

i. EPCAM/CD49f Analysis (7-AAD, Lineage PE cocktail, CD49f-FITC andEPCAM-APC)

The cell number can be reduced in future experiments once the detectors, gates, andcompensation values have been determined.

15. Centrifuge the cells for 5 min at 400 × g, 4◦C.

16. Aspirate the supernatant and carefully resuspend the pellet with 200 μl of HBSScontaining 2% FBS.

17. Add the appropriate antibodies to tubes b, c and e to i in step 14 according to thedilutions listed in Table 14.23.3.

Alternatively, use a stock solution of all eight PE-conjugated antibodies to resuspend thesamples requiring the lineage cocktail in step 16.

18. Incubate the cells for 30 min at 4◦C.

19. Add 1 ml of HBSS containing 2% FBS to each tube.

20. Centrifuge the cells for 5 min at 400 × g, 4◦C. Wash the cells by aspirating thesupernatant and carefully resuspending the pellet with 1 ml of HBSS containing 2%FBS.

21. Centrifuge the cells for 5 min at 400 × g, 4◦C. Aspirate the supernatant and carefullyresuspend the pellet with 300 μl of HBSS containing 2% FBS.

22. Add 5 μl of 7-AAD (0.25 μg/1.0 × 106 cells; see Table 14.23.3) to samples d, f, andi.

23. Pass the cell suspension through a 100-μm strainer and transfer to a FACS tube

24. Incubate for 15 min at 4◦C.

FACS analysis and gating procedure25. Analyze tubes a to e, g, and h by flow cytometry (Coligan et al., 2012, Chapter 5) to

set up the gates, detector voltages, and compensation.

26. Analyze samples f and i and use the sequential gates described below. Examples ofthe FACS plots and gating procedure are illustrated in Figure 14.23.4A and B.


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a. SSC versus FSC: eliminate debris and cell clusters.

b. FL3 (7-AAD) versus FSC: gate only live cells (7-AAD negative).

c. FL2 (PE) vs. FSC: gate only lineage negative cells (PE negative).

d. FL4 (APC) vs. FL1 (FITC): determine either the percent of CD44Hi/CD24Low forsample f or EPCAMHi/CD49fLow for sample i.

BASICPROTOCOL 4

SUBCUTANEOUS IMPLANTATION OF ESTROGEN PELLETS IN MICERECEIVING ESTROGEN RECEPTOR–POSITIVE TUMORS

In the clinic, breast cancer is subtyped by pathological and molecular analyses intoestrogen receptor (ER)-positive and -negative tumors. ER status provides prognostic in-formation and guides therapeutic options for patients. Importantly, ER+ breast cancersoften require systemic estrogen for growth in vivo. It is important to provide supplemen-tal estrogen to mice receiving ER+ tumors (supplementation is not necessary for ER-breast cancer tissue). The ER status of breast cancer samples can be obtained from thepathology report of the patient. If a pathology report is not available, a small portionof the patient’s tumor specimen should be analyzed for ER status [by paraffin embed-ding, sectioning and immunohistochemistry against ER (DeRose et al., 2011)] while theremaining tissue can be cryopreserved, preferably as fragments. Once the ER status isverified, the cryopreserved tissue can be transplanted into mice. It is best to use high-doseestrogen pellets that provide adequate estrogen supplementation for up to 1 year (Rudaliet al., 1975), although we typically replace these pellets at 6 months.

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% ethanolPovidone-iodine (betadine) swabsticks (widely available)Estrogen pellets, stored on ice (see Support Protocol 3 for preparation of pellets)Lidocaine-bupivacaine mixture (see recipe)

Dissecting scissors, sterileForceps, sterileHot 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 boardAnesthetic vaporizer (VetEquip, cat. no. 911103)Compressed oxygenAnesthesia induction chamber (VetEquip, cat. no. 941444)Label tapeCotton swabs, sterile

1. Autoclave surgical tools prior to the procedure and disinfect in between animals usinga hot bead sterilizer. Disinfect the surgical bench area by wiping with a disinfectantsuch as CLIDOX-S. Turn on the heating pad and set the water temperature to 38◦C.

2. Using an induction chamber, anesthetize the mouse by exposure to 2% to 2.5%vaporized isoflurane in oxygen, then transfer the animal to the surgical platform


14.23.16


surgically remove

peritoneum

“clearedmammary fat pad”with transplanted

tissue

hormone pelletimplantation site(subcutaneous)

lymph node

nipple

nipples

1.2 cm1.2 cm

1.5 cmincisions

blood vessel

site of tissuetransplantation

Figure 14.23.5 Schematic showing location of mouse mammary fat pads, surgical incisions, and sites fortissue/cell transplants and the estrogen pellet implant. Mouse diagram was adapted from Hummel et al. (1966)with permission of The Jackson Laboratory.

and place it in a prone position. Stabilize the mouse by gently fixing its legs tothe platform with tape, making sure that the nose cone is secured for continuousdelivery of vaporized isoflurane. Remove the fur from the surgical area betweenthe shoulder blades (approximately 6 × 6–mm) by applying a depilatory cream andgently removing the hair with a cotton swab.

3. Pinch the footpad to make certain the mouse is in the proper plane of anesthesiabefore surgery. If the animal responds to the pinch by twitching, allow more time forthe anesthesia to take effect.

4. When the mouse is fully anesthetized, disinfect the skin with 70% ethanol and wipethree times with betadine using a swab stick.

5. Make a small (3-mm) incision between the shoulder blades (see Fig. 14.23.5), beingcareful not to cut any muscle. Make a subcutaneous pocket for the estrogen pelletusing blunt dissection technique. Apply lidocaine-bupivacaine mixture to the exposedtissue.

6. Insert one pellet into the pocket.

7. Close the incision with a wound clip.

8. Proceed to breast tumor transplantation (Basic Protocol 5).

BASICPROTOCOL 5

IMPLANTATION OF BREAST TUMOR TISSUE FRAGMENTS INTO MICE

This is a subcutaneous procedure that does not require laparotomy. We perform thisprocedure on mice that are 3 to 4 weeks of age to facilitate simultaneous clearing ofthe immature endogenous mouse mammary tissue (a video demonstrating clearance ofmouse mammary fat pads for tissue transplantation can be found at: http://www.jove.com/video/1849/mammary-epithelial-transplant-procedure). It is also possible to pre-clear


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mammary tissue at 3 to 4 weeks, followed by a second surgery to implant tumor cells ata later date. All tumors are implanted into the inguinal (4th) mammary fat pad (Hummelet al., 1966, and Fig. 14.23.5). We occasionally implant tumors into both contralateral fatpads if the goal is to amplify tumor tissue. We use the same procedure for NOD/SCID(The Jackson Laboratory, stock #1303) and NOD/SCID/IL2Rγ -/- (NSG) mice (TheJackson Laboratory, stock #5557). Surgery should not be performed on the same day themice are transported from another facility. If mice are shipped from a vendor, we allowthem to acclimatize for 4 to 7 days prior to surgery.

Materials

Tumor samples for implantation fresh or frozen (see Basic Protocols 1 and 2)NOD/SCID mice (The Jackson Laboratory, stock #1303); alternatively, the more

immune-compromised NOD/SCID/IL2Rγ -/- (NSG) mice (The JacksonLaboratory, stock #5557) can be used

HBEC medium (see recipe)70% ethanolClidox−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 tubes50-ml conical tubes, sterileHeating pad (water circulating)Dissecting scissors, sterileForceps, sterileSterile 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 ScienceTools)

Hot bead sterilizer (Fine Science Tools, cat. no. 18000-45)Surgical platform, such as a foam boardAnesthetic vaporizer (VetEquip, cat. no. 911103)Anesthesia induction chamber (VetEquip, cat. no. 941444)9-mm nose cone (VetEquip, cat. no. 921609)Label tapeCotton swabs, sterileTerumo U-100 insulin syringes with 27-G × 1/2 in. needles, sterileBattery-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 (Basic Protocol 1)

1a. For fresh tissue: Prepare 4 × 2–mm tumor tissue fragments as described in BasicProtocol 1. Place fresh tissue fragments in a 1.5-ml sample tube with ∼ 0.5 ml sterileHBEC medium (alternatively, DPBS, DMEM/F12, or RPMI can be used) to keep thetissue moist. Place the tube on ice until implantation.

1b. For cryopreserved tissue: Thaw frozen tissue fragments by placing the vial in a 37◦Cwater bath and removing before the medium completely melts. Spray the outsideof the vial with 70% ethanol, then move the vial into a biosafety hood and placethe contents in a 50-ml conical tube. Add 10 ml of HBEC medium, then aspirateto remove the DMSO-containing freezing medium. Repeat three times. Add freshHBEC medium, close the lid, and place the tube on ice until the implantation.


14.23.18


2. Set up the surgical suite. Turn on the heating pad and set the water temperature to38◦C. Autoclave surgical tools at the beginning of the procedure and disinfect inbetween animals using a hot bead sterilizer. Disinfect the surgical bench area bywiping with a disinfectant such as Clidox−S.

3. Anesthetize the mouse by exposure to 2% to 2.5% vaporized isoflurane in oxygenusing an induction chamber, then transfer it to the surgical platform and place it in asupine position. Stabilize the mouse by gently fixing its legs to the platform with tape,making sure the nose cone is in place for continuous delivery of vaporized isoflurane.Denude the surgical area by applying a depilatory cream and gently removing thehair with a cotton swab.

4. Pinch the footpad to make certain the mouse is in the proper plane of anesthesia. Ifthe animal responds to the pinch by twitching, allow more time for the anesthesia totake effect. When the mouse is fully anesthetized, disinfect the skin with 70% ethanoland wipe three times with betadine using a swab stick.

5. Make two small subcutaneous incisions in the skin to expose the fat pad: one alongthe midline (approximately 1.5 cm) and one a short distance down the leg (about1.2 cm; Fig. 14.23.5). Apply lidocaine/bupivicaine anesthetic mixture to the incisionusing a swab stick.

Ensure that the incisions are not too close to the genitals, since the wound clips mayimpede their function. Take care not to puncture the peritoneal membrane or to cut thevessels in the leg. There should be little or no bleeding with this procedure.

6. Expose the mammary fat pad using a sterile cotton swab, and pin the skin down usinga small needle. Locate the inguinal lymph node in the middle of the fat pad (it is palerthan the surrounding fat pad, has a kidney bean shape and is located at the junctionof blood vessels).

Using the lymph node as a reference for the center of the gland, the site of transplantationis in the center of the half of the fat pad that is proximal to the peritoneum (see Fig.14.23.5).

7. Insert the tip of a cross-action tweezers into the center of the transplantation site andmake a pocket about 3 mm long. Place a tumor fragment into the pocket. Carefullyremove the tweezers once the tissue has been placed.

8. Clear the mammary fat pad by using forceps and scissors. Grasp the portion of fat padclosest to the nipple and cut under the fat pad with the scissors, keeping the scissorsparallel and adjacent to the skin. Continue cutting the fat pad along the skin until thelymph node is reached. Cut across the width of the fat pad immediately adjacent (onthe peritoneal side) to the lymph node, and remove this fat pad tissue to clear thegland. Be careful not to disrupt the pocket containing the tumor fragment.

Some bleeding may occur as the fat pad is cleared. It is advisable to have a battery-operatedcautery unit on hand to stop bleeding if necessary. If the #5 mammary fat pad is attachedor in close proximity to the cleared #4 fat pad, the endogenous mammary epithelium fromthe #5 gland will invade the cleared #4 fat pad. Removal of any #5 mammary tissue thatis close or attached to the cleared fat pad will prevent this from occurring.

9. Close the incision with wound clips. Place the mouse on the warm pad until itawakens. Return the mouse to its home cage.

10. Monitor the mice according to the approved IACUC protocol—we monitor the micedaily for 3 to 5 days post-surgery and remove wound clips within 7 to 10 days. Checktumor growth weekly and determine and record its size with the caliper.

If the mice show any sign of discomfort or illness, a veterinarian should be consulted. Ifthe tumor reaches 2 cm in diameter or ulcerates, the mice should be humanely euthanized.


14.23.19


BASICPROTOCOL 6

TRANSPLANTING TUMOR CELLS AND ORGANOIDS INTO MICE

Organoids and cells isolated from digestion of solid tissue, or separated from body fluid(using procedures described in Basic Protocols 1 and 2), can be injected into clearedmouse fat pads to generate tumor grafts. We typically inject cells and organoids withMatrigel to facilitate their growth.

Materials

Organoids or cells from Basic Protocol 1 or 2Growth factor–reduced Matrigel (BD, cat. no. 354230; or see Support Protocol 3

for preparation of EHS matrix)NOD/SCID mice (The Jackson Laboratory, stock #1303); alternatively, the more

immunocompromised NOD/SCID/IL2Rγ -/- (NSG) mice (The JacksonLaboratory, 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 intomice (Basic Protcol 5)

1. Thaw the Matrigel at 4◦C and keep it cold at all times to prevent solidification.Resuspend the organoids or cells in Matrigel. Adjust the concentration to 1–2 ×106 cells in 20 μl Matrigel for each intended injection. Keep tube with Matrigel/cellmixture on ice at all times.

When not using the syringe, keep it on ice to prevent the Matrigel from solidifying prior toinjection. If 10 fat pads are to be injected, prepare sufficient suspension for 11 injections.For this number, resuspend 11–22 × 106 cells in 220 μl of Matrigel to compensate formaterial loss on the tube and syringe.

2. Inject the cell/Matrigel mixture into mammary fat pads using a similar procedure tothat described in Basic Protocol 5, with the exception that a tissue pocket is not used.Instead, inject 20 μl Matrigel/cell mixture directly into the mammary fat pad usingan insulin syringe with a 27-G × 1/2 in. needle.

The mammary gland is then cleared, and the mice are closed and monitored as describedin Basic Protocol 5.

BASICPROTOCOL 7

HARVESTING TUMORGRAFTS FROM MICE

If the tumor cells are not fluorescent or otherwise labeled, necropsy must be performedvery carefully to identify potential sites of metastasis. Tumor and metastasis samplesmust be kept on ice to help maintain viability as well as prevent degradation of protein,DNA, or RNA. Store the tissue samples in DPBS to prevent drying while harvestingother tissues. Keep the 4% PFA on ice with the cap closed, or wrap the opening withparafilm to prevent PFA vapor from escaping. Separate the tools used for fixatives fromthose used for live tissue to prevent cross contamination.

Materials

70% ethanolTumor-bearing mouse (see protocols above)4% paraformaldehyde (PFA; APPENDIX 3D)Liquid nitrogenTissue freezing medium (see recipe)

Dissecting equipmentCotton swabsCalipers for measuring tumorBalance for weighing tumor


14.23.20


Plastic petri dish (Fisher Scientific, cat. no. 08-757-14)15- and 50-ml conical tubes, sterile1- to 1.5-ml cryovials, sterileHyclone DMEM/F12 (1:1) with HEPES (Thermo Scientific, cat. no. SH30023.01)

Additional reagents and equipment for CO2 asphyxiation of the mouse (Donovanand Brown, 2006) and freezing of samples (Basic Protocol 1, “b” steps)

1. Clean the workbench and all equipment with 70% ethanol.

2. Euthanize the mouse by CO2 asphyxiation following IACUC-approved procedures(Donovan and Brown, 2006).

3. Lay the mouse in a supine position on the surgical platform and clean the incisionarea with 70% ethanol.

4. Make an incision to expose the tumor. Carefully detach the skin from the tumor usingcotton swabs and scissors.

5. Measure the tumor (length × width × height) and record (it is easier to measure thetumor before isolating it from the skin). Carefully remove the tumor.

6. Record the wet weight of the tumor.

7. When harvesting the tumor: Put the tumor sample in a petri dish and place it on ice.Divide the tumor into portions for different applications, such as histology, nucleicacid, and protein isolation, and generation of viable organoids or cells. Process thetissue as described in steps 9 to 12.

Portions do not need to be equally divided, but should be a size most appropriate for thesubsequent application. For example, histology can performed on small pieces dissectedfrom several different regions of the tumor, while protein and viable cell preparations needsignificant amounts of starting tissue.

8. For metastases: Look for metastases by carefully examining the axillary lymph nodes,lungs, intestines, spleen, liver, kidneys, ovaries, uterus, and other lymph nodes. Lookfor abnormal nodules in soft tissue, enlarged organs, and enlarged lymph nodes (2 mmor larger) as potential indicators of metastasis. Place organs with potential metastasisin a petri dish on ice. Process the tissue as described in steps 9 to 12.

Micrometastases and bone metastases are usually not grossly observable. Thus, moresensitive methods, such as histology, must be used to determine if micrometastases arepresent.

If the metastasis is large enough, samples can be processed for histological analysis(to confirm the metastasis) and any remaining tissue can be used for transplantation asfragments. Photograph the samples to record the gross abnormality suspected to be ametastasis.

9. For histological analysis: Fix tissue samples in 2 to 5 ml of cold 4% PFA in a 15-mlconical tube. Keep the sample tubes on ice until they are ready to be stored in therefrigerator (fixation is typically overnight at 4◦C). Move the samples to 70% ethanoland store at 4◦C no longer than 1 week prior to embedding in paraffin. Section andstain the tissue as desired.

10. For nucleic acid and protein isolation: Place the samples in a cryovial or wrap inaluminum foil and submerse in liquid nitrogen to flash freeze. Transfer the tissue to a−80◦C freezer. Isolate nucleic acids or protein using any of several standard methods.

11. For transplantation: Cut the tumor tissue into approximately 4 × 2–mm fragmentsand freeze in tissue freezing medium (do not confuse with HBEC freezing medium)in cryovials. Freeze at −80◦C overnight as described in Basic Protocol 1, “b” steps,


14.23.21


and then store in liquid nitrogen. Transplant the tissue as described in Basic Protocol5.

12. For processing into viable cells: Place the solid tumor tissue (size can vary) in a50-ml conical tube containing sterile DMEM/F12 medium. Keep the tissue on iceuntil processing can be performed as described in Basic Protocol 1.

BASICPROTOCOL 8

PRODUCING AND UTILIZATION OF LENTIVIRUSES FORTRANSDUCTION OF TUMOR CELLS

Bioluminescence and/or fluorescence imaging techniques are useful for monitoring tumorgrowth in live animals and to locate metastases. However, tumor cells derived frompatients and tumorgrafts can have low transfection or transduction rates when standardprocedures are employed. Therefore, concentrated lentiviruses are utilized, since theyare able to effectively transduce primary cells. This protocol describes the productionand titering of a concentrated lentivirus, which co-expresses both ZsGreen and luciferase(pHIV-Luc-ZsGreen), for the purpose of labeling cells to identify micrometastases intumorgrafts. In addition, a suspension infection method adapted from Welm et al. (2008),which is used to transduce primary cells, including mixed adherent/suspension cultures,is described below.

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 bleachHyclone 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)

Hemacytometer15- and 50-ml conical centrifuge tubes (e.g., BD Falcon)Centrifuge with swinging-bucket rotor, refrigerated


14.23.22


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)

BalanceUltracentrifuge (Beckman Coulter Optima LE80k with swinging-bucket SW28

rotor)0.5-ml microcentrifuge tubes6-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., 2012, Chapter 5)Light microscope

Additional reagents and equipment for counting cells using a hemacytometer(Strober, 1997), flow cytometry (Coligan et al., 2012, Chapter 5), andpreparation of organoids (Basic Protocol 1)

Prepare concentrated lentiviral particlesNOTE: All protocols using lentiviruses must be reviewed and approved by an InstitutionalBiosafety Committee. BSL2 techniques are employed when generating and utilizinglentiviruses.

1. Expand HEK 293T cells in DMEM medium supplemented with 10% FBS (heat inacti-vated) in 10-cm tissue culture dishes. Culture the cells at 37◦C in a 5% CO2 humidifiedincubator.

For unknown reasons, high-passage HEK 293T cells do not produce high-titer lentivirus.It is best to obtain a fresh stock of HEK 293T from the ATCC (#CRL-11268) rather thanacquire stocks from other laboratories. Also, cryopreserve early-passage cells and thawa fresh stock for the generation of high-titer lentivirus.

2. Aspirate the medium and wash each plate with 5 ml of DPBS.

3. Add 1 ml/plate of 0.05% trypsin-EDTA and incubate for 2 to 5 min. Gently pipet upand down to mix the trypsin solution.

4. Add 10 ml/plate of DMEM medium supplemented with 10% FBS (heat-inactivated)and determine the number of cells per ml using a hemacytometer (Strober, 1997).

5. Plate 4–7 × 106 cells into each 10-cm tissue culture dish and adjust the vol-ume of each plate to 10 ml with DMEM medium supplemented with 10% FBS(heat inactivated). Culture the cells overnight at 37◦C in a 5% CO2 humidifiedincubator.

Alternatively, a confluent 10-cm plate of HEK293T cells can be replated at a 1:10 dilutionand cultured for 2 to 5 days until ∼90% confluent. A typical procedure utilizing 24 10-cmplates yields ∼0.9 to 1.2 ml of a solution containing between 5.0 × 107 and 5.0 × 108

infectious units/ml (IU/ml).

6. Aspirate the medium, add 8 ml/plate of DMEM medium supplemented with 10%FBS (heat inactivated) that has been prewarmed to 37◦C, and culture for 2 hr.

7. Prepare PEI at 1 mg/ml (w/v) in deionized sterile water.

The transfection procedure described in this protocol is optimized for PEI (Huh et al.,2007). The PEI solutions can be aliquotted into 1.5-ml microcentrifuge tubes and stored


14.23.23


at −20◦C for up to 1 year. If a different transfection reagent will be used, optimizationwill be necessary.

8. Add 720 μl of the 1 mg/ml PEI solution to 11.28 ml of OPTI-MEM medium in a50-ml conical tube, which yields a solution sufficient to transfect 24 plates (30 μg ofPEI per plate in 500 μl of OPTI-MEM).

The volume can be adjusted depending on the number of plates utilized.

9. Prepare a solution of plasmids in a separate 15-ml conical tube, which is enough totransfect 24 plates, by adding 120 μg of pHIV-Luc-ZsGreen (5 μg/plate), 40.8 μgof pMDLg/pRRE (1.7 μg/plate), 40.8 μg of pRSV-Rev (1.7 μg/plate), and 40.8 μgof pCMV-VSV-G (1.7 μg/plate) to a final volume of 12 ml of OPTI-MEM medium(500 μl/plate).

It is important to utilize plasmid solutions at >0.5 μg/μl, since lower concentrations candilute the medium/DNA solution and decrease the transfection efficiency. In addition, toachieve high viral titers, it is necessary to mix all four plasmids together before mixingwith the PEI solution.

10. Add 12 ml of the plasmids/OPTI-MEM solution (prepared in step 9) to the 12 ml ofPEI/OPTI-MEM (prepared in step 8) and mix. Incubate the solution for 30 min atroom temperature.

11. Add 1 ml/plate dropwise of the PEI/plasmid/OPTI-MEM solution from step 10 to theplates from step 6, gently swirl the plates, and culture cells with solution for 24 hr at37◦C in a 5% CO2 humidified incubator.

12. From this point forward, utilize BSL2 procedures according to your institution’sbiosafety protocol. Every time before working with solutions containing virus, placea large beaker containing >50 ml of bleach in a biosafety cabinet to disinfect anymedium and pipets (make sure the final concentration of bleach is >10% beforeremoving it from the biosaftey cabinet).

The use of personal protective equipment, such as double gloves, arm sleeves, face masks,eye protection, laboratory coats, etc., is required. Tubes containing virus can only beopened in a biosafety cabinet. Sealed tubes must be sprayed with an 80% ethanol solutionbefore removal from the biosafety cabinet.

13. Remove the medium from each plate and transfer it to the beaker containing bleach.Add 8 ml/plate of DMEM medium containing 10% FBS (heat inactivated) that hasbeen pre-warmed to 37◦C and incubate overnight at 37◦C in a 5% CO2 humidifiedincubator.

14. The following day (48 hr post transfection), remove the medium from each plateand transfer it to a 50-ml conical tube on ice. Immediately add 8 ml/plate of freshprewarmed DMEM medium containing 10% FBS (heat inactivated) to each plate andculture overnight at 37◦C in a 5% CO2 humidified incubator.

The transfection efficiency can be estimated at this stage by using a tissue culture mi-croscope with fluorescence capability and observing the percent of cells fluorescent forZsgreen. It is advisable that this be performed on an extra plate of cells that are fixed with4% paraformaldehyde, to inactivate the lentivirus, prior to microscopy. Continue with theconcentration procedure only if >50% of the cells are fluorescent. If <50% of the cellsare transfected, optimize the transfection procedure before proceeding.

15. Centrifuge the 50-ml conical tubes containing the medium for 10 min at 2500 × g at4◦C to pellet any large debris.

16. Pass the supernatant through a 0.45-μm cellulose acetate filter and transfer 32 ml(about four plates worth) to each of six ultracentrifuge tubes. Place tubes in titanium


14.23.24


buckets on ice. Balance bucket pairs by adjusting the weight with DMEM medium(this is done in the biosafety cabinet until the buckets are sealed).

17. To pellet the virus, ultracentrifuge the solution for 1 hr 45 min at 112,000 × g, 4◦C,using an SW28 swinging-bucket rotor.

18. Carefully remove the supernatant and transfer it to a beaker containing bleach. Im-mediately (do not let the pellet dry) add 150 μl of DMEM/F12 medium to eachcentrifuge tube on ice. Gently pipet up and down without creating bubbles to dissolvethe pellet. Place the ultracentrifuge tubes in a 50-ml conical tube and cap the conicaltube to prevent evaporation of the solution. Store the conical tubes at 4◦C overnight.

19. The following day, collect the second round of medium from the plates (72 hr posttransfection) and process it in the same manner as in steps 14 to 17.

Add bleach at a final concentration of 10% to the plates, incubate at room temperaturefor 20 min in the biosafety cabinet, and discard plates and medium.

20. After the ultracentrifugation in step 17, remove the supernatant. Resuspend the pelletsby transferring the DMEM/F12 solution containing the concentrated virus that wascollected 48 hr post transfection to each tube. Gently pipet up and down to dissolve thepellet. Place the ultracentrifuge tubes in a 50-ml conical tube to prevent evaporation,and incubate at 4◦C for 2 hr to ensure the virus is completely dissolved.

21. Pipet up and down to resuspend the pellets, being careful to limit bubbles. Combineall of the resuspended pellets into one tube and gently pipet to mix. Aliquot 50 to 100μl of the concentrated virus into 500-μl microcentrifuge tubes and store at −80◦C.

The virus solutions are aliquotted to minimize freeze/thaws, which can significantly reducethe number of IU/ml. The virus solutions can be stored up to 1 year at −80◦C.

Determine infectious units (IU)/ml by FACS22. Expand HEK 293T cells in DMEM medium supplemented with 10% FBS (heat-

inactivated) in 10-cm tissue culture dishes. Culture the cells at 37◦C in a 5%CO2 humidified incubator.

23. Aspirate the medium and wash each plate with 5 ml of DPBS.

24. Add 1 ml/plate of 0.05% trypsin-EDTA and incubate for 2 to 5 min at 37◦C in a 5%CO2 humidified incubator. Gently pipet up and down to mix the trypsin solution.

25. Add 10 ml/plate of DMEM medium supplemented with 10% FBS (heat inactivated)and determine the number of cells per ml using a hemacytometer (Strober, 1997).

26. Plate 2.0 × 105 cells in each well of a tissue-culture-treated 6-well tissue culture plateand adjust the volume to 3 ml/well with DMEM medium supplemented with 10%FBS (heat inactivated). Culture the cells overnight at 37◦C in a 5% CO2 humidifiedincubator.

27. Aspirate the medium from four wells of the 6-well plate and add 1 ml/well of DMEMmedium supplemented with 10% FBS (heat inactivated) that has been prewarmed to37◦C.

28. Aspirate the medium from the remaining two wells of the 6-well plate and carefullywash with 2 ml/well of DPBS so the cells do not detach.

29. Add 0.5 ml of 0.05% trypsin-EDTA to each of the two wells and incubate for 2 to5 min at 37◦C in a 5% CO2 humidified incubator. Gently pipet up and down to mixthe trypsin solution.


14.23.25


30. Add 0.5 ml/well of DMEM medium supplemented with 10% FBS and determine thenumber of cells present in each well independently using a hemacytometer (Strober,1997). Calculate the average number of cells present in each of the two wells.

This number will be used to determine the IU/ml.

31. From this point forward, utilize BSL2 procedures according to your institution’sbiosafety protocol. Thaw an aliquot of concentrated virus in the biosafety cabinet.

32. Dilute the virus solution 1:10 in DMEM medium supplemented with 10% FBS (heatinactivated). For example, add 8 μl of virus solution to 72 μl of medium.

33. Add different amounts of the diluted virus to each of the remaining four wells asdescribed below, gently mix and culture overnight.

a. No-virus control.

b. 1 μl of diluted virus (1:10,000 final dilution).

c. 10 μl of diluted virus (1:1000 final dilution).

d. 50 μl of diluted virus (1:200 final dilution).

The final dilution values are based upon the use of a 1:10 initial dilution of the virus and1 ml of medium in each well. Different dilutions are required, since a sample containing<15% ZsGreen positive cells determined by FACS is required to more accurately calculatethe IU/ml.

34. Remove the medium 24 hr post infection and put into bleach. Add 3 ml/well ofDMEM medium supplemented with 10% FBS (prewarmed to 37◦C) and culture for2 additional days.

35. Remove the medium 72 hr post infection and put into bleach. Carefully wash thewells with 2 ml/well of DPBS so the cells do not detach.

36. Add 0.5 ml/well of 0.05% trypsin-EDTA and incubate for 2 to 5 min. Gently pipetup and down to mix the trypsin solution.

37. Transfer the cell suspension to a 15-ml conical tube on ice. Wash each well with 1ml of HBSS containing 2% FBS and transfer this solution to the respective 15-mlconical tube.

38. Add paraformaldehyde to a final concentration of 2% to each tube and incubate at4◦C for 20 min to inactivate any residual virus that may be present.


40. Aspirate the supernatant and carefully resuspend the pellet with 1 ml of HBSScontaining 2% FBS.


42. Aspirate the supernatant and carefully resuspend the pellet with 250 μl of HBSScontaining 2% FBS.

43. Pass the cell suspension through a 100-μm cell strainer and transfer to a FACS tube.

44. Set up the gates and detector voltages, and analyze samples by flow cytometry usingthe sequential gates described below (Coligan et al., 2012, Chapter 5).

a. SSC versus FSC: eliminate debris and gate only single cells.

b. FL1 (ZsGreen) histogram: determine percent ZsGreen-positive cells.

45. Calculate the IU/ml using the formula below.


14.23.26


IU/ml = (average # of cells/well) * (% ZsGreen positive cells/100) * (virusdilution)

The average number of cells/well was determined above in step 30. We typically use adilution where the cells are <15% ZsGreen positive. Titers ranging from 5 × 107 to 5 ×108 IU/ml are commonly achieved using this protocol.

Transduce primary cells46. Prepare organoids as described in Basic Protocol 1.

47. Resuspend the organoids in a 1:1 mixture of 0.05% trypsin-EDTA and DPBS. Placethe sample into one well of a 6-well plate (any type) to facilitate viewing under themicroscope.

We typically use 1 ml of diluted trypsin solution for each 0.5 g of dissected tumor tissue(measured prior to collagenase digestion).

48. Trypsinize the tissue at room temperature, and carefully monitor the organoid disso-ciation process every few minutes under low magnification using a light microscope.Trypsinize for 1 min, then very gently pipet the cells up and down with a 1-ml pipetto help dissociate the organoids into single cells. Repeat every minute until most ofthe organoids are dissociated.

49. Once the organoids are dissociated, immediately add 5 ml HBEC medium (orDMEM/F12 containing 10% FBS) to neutralize the trypsin, and centrifuge the cells 5min at 530 × g, room temperature. Wash cells with 5 ml medium two additional times.Resuspend the pellet in HBEC medium and count the cells using a hemacytometer(Strober, 1997).

If the pellet is difficult to resuspend because of viscosity, it is likely due to the presence ofDNA from cells that lysed. In this case, incubate the cells with 10 μg/ml DNase I (finalconcentration) in HBEC medium for ∼5 min at room temperature.

50. Seed 250,000 cells per well in a 6-well plate in 0.5 ml of HBEC medium.

For suspension infections, use ultra-low attachment plates, and for monolayer infections,use regular tissue culture-treated plates. The best method of infection must be determinedempirically for each tumor. For monolayer infections, allow the cells to attach for 24 to48 hr prior to adding virus.

51. Add polybrene to a final concentration of 1 to 4 μg/ml (the actual concentrationshould be empirically determined using the target cells). Add concentrated lentivirususing a multiplicity of infection (MOI) of 5 to 20 viral particles/target cell. Addadditional HBEC medium to bring the final volume to 1 ml per well.

The MOI may also have to be optimized for different tumors.

52. Centrifuge the plate 1 hr at 700 × g, room temperature. Alternatively, proceed di-rectly to step 54 without performing the centrifugation, replacing the virus-containingmedium with fresh HBEC medium after overnight incubation.

Some cells are more efficiently infected using a combination of the spin method andovernight suspension culturing with virus. This can only be determined empirically.

53. Remove the virus medium and add 2 ml of fresh HBEC medium.

54. Incubate at 37◦C in 5% CO2 humidified incubator—expression of virus proteins (e.g.,ZsGreen) should be detectable by fluorescence microscopy after 72 hr. Feed the cellsevery day by exchanging the medium.

55. Expand the cells until a sufficient number is obtained to transplant the desired numberof mouse mammary fat pads (see Basic Protocol 6).


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During culture, some of the tumors attach to tissue culture-treated dishes, some grow insuspension, and some are mixed. To propagate suspension or mixed culture, centrifugethe solution containing cells 5 min at 530 × g, room temperature, aspirate the medium,suspend the cells in 1 ml of HBEC medium, and add them with fresh HBEC medium tothe adherent population. Adherent cells can be collected by first washing the plate with5 ml of DPBS followed by addition of 1 ml of 0.05% trypsin-EDTA. Once the cells detach,the resulting suspension can be mixed with the suspended cells, if applicable. Feed thecells every day by aspirating the medium and adding fresh medium for adherent cells.For suspension cells, follow the procedure described above for propagation. We haveonly attempted to maintain primary tumor cells in culture for up to 2 weeks using theseconditions.

BASICPROTOCOL 9

THREE-DIMENSIONAL PRIMARY TUMOR CULTURES

The physical organization of breast cancer in three dimensions (3-D) modulates signalingpathways that affect the survival, growth, and sensitivity of tumor cells to therapeutics.Primary tumor organoids can be grown in 3-D culture to study the complex cell-celland cell-matrix interactions that occur in vivo. Every primary human or xenograft tumormay require different medium supplements for optimal growth. A standardized mediumis typically used to simplify in vitro culturing conditions. The medium that we use is amodified version of the M87 medium developed by the Stampfer laboratory that supportsthe growth of human primary mammary epithelial cells (Garbe et al., 2009). We havecultured several primary and xenograft tumor cells successfully for up to 2 weeks usingthis medium, although additional optimization may be possible.

Materials

Vial of cryopreserved human primary tumor cellsModified M87 medium (see recipe)Organoids, dissociated tumor cells, or pleural effusion cells (see Basic Protocols 1

and 2)Growth factor-reduced Matrigel (see recipe)Test compoundsVehicle (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)CentrifugeLight microscope55-ml disposable reagent reservoir (Bioexpress, cat. no. B-0812-7)96-well untreated clear U-bottom plates (BD Falcon, cat. no. 351177)12-channel pipettor50-ml multichannel pipetting reservoir96-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 nmConfocal microscope

Additional reagents and equipment for counting cells using a hemacytometer(Strober, 1997)


14.23.28


Defrost cryopreserved tumor cells (if applicable)1. Defrost a vial of cryopreserved human primary tumor cells by submerging it in a

37◦C water bath for 2 to 3 min. As soon as the vial is thawed, transfer the contentsto a 15-ml conical tube containing 10 ml of modified M87 medium that has beenprewarmed to 37◦C.

2. Centrifuge the cells for 5 min at 700 × g, room temperature.

3. Aspirate the supernatant and carefully resuspend the pellet into 1 ml of modifiedM87 medium by pipetting up and down. Perform a trypan blue exclusion test usinga hemacytometer to determine the number of viable cells per ml (Strober, 1997).

Aggregate single tumor cells (if applicable)Typically, organoids are used for the 3-D in vitro assay. Organoids prepared as de-scribed in Basic Protocol 1 can be embedded directly in Matrigel. Dissociated tumorcells and pleural effusion cells (Basic Protocol 2) should be aggregated prior to em-bedding in Matrigel. However, some pleural effusion cells do not aggregate under theseconditions.

4. Suspend the single cells in modified M87 medium to achieve a concentration of 1–4× 106 cells/ml.

5. Add 1 ml of tumor cell suspension to each well of a 24-well ultra low-adhesion plate.Incubate the cells at 37◦C in a 5% CO2 humidified incubator overnight.

6. Separate tumor aggregates from single cells by differential centrifugation. Placeaggregates in a 15-ml conical tube with 5 ml of modified M87 medium and centrifugefor 30 sec to 1 min at 450 × g, room temperature (single cells will remain in suspensionwhile cellular aggregates pellet). Remove the supernatant and resuspend the pelletin 5 ml of modified M87 medium. Monitor the organoid enrichment procedureby analyzing 25-μl samples of the resuspended pellet by light microscopy. Repeatcentrifugation process two to five times as needed to enrich for aggregated tumorcells.

Embed tumor organoids or aggregates in Matrigel7. Defrost a bottle or aliquot of frozen growth factor–reduced Matrigel at 4◦C.

This process can take several hours to overnight. It is very important to keep the Matrigelat 4◦C at all times to prevent it from solidifying.

8. Carefully transfer the organoids/aggregates to a 15-ml conical tube containing 10 mlof the modified M87 medium that has been prewarmed to 37◦C. Centrifuge the cellsfor 30 sec at 400 × g at room temperature. Aspirate the supernatant to remove thesingle cells. Resuspend the pellet by slowly adding 1 ml of modified M87 medium,taking care to avoid breaking up the organoids. Perform a trypan blue exclusion testusing a hemacytometer to determine the number of viable organoids/aggregates perml (Strober, 1997).

9. Centrifuge the organoids/aggregates 5 min at 400 × g, room temperature. Removethe supernatant and place the tube on ice. Determine the number of organoids andthe volume of Matrigel to be added to each well of a 96-well plate.

For a viability assay using MTS as the endpoint, 300 to 500 organoids are typicallyembedded in 30 to 40 μl of Matrigel per well of a 96-well U-bottom clear, non-tissue-culture-treated plate. U-bottom plates are used to minimize the area of Matrigel touchingthe plastic to reduce the number of tumor organoids adhering to the plate. To acquirehigh-resolution images using confocal microscopy with a live/dead readout, 100 to 300organoids are usually embedded in 50 to 100 μl of Matrigel per well of a 96-well clear-bottom, black wall plate.


14.23.29


10. Based upon the experiment (MTS or Live/Dead), determine the total number ofwells and amount of Matrigel required to achieve the proper concentration oforganoids/aggregates per ml. Carefully suspend the pellet of organoids/aggregatesin Matrigel on ice.

11. Transfer the Matrigel cell suspension to a 50-ml disposable reservoir on ice. Using a12-channel pipettor, carefully mix the suspension by gently pipetting up and down. Ifperforming an MTS endpoint assay, add 30 to 40 μl of the cell suspension/Matrigelmixture to each well of the 96-well plate. If performing a live/dead assay, add 50 to100 μl of the Matrigel/cell suspension to each well of the 96-well plate.

Care should be taken to avoid the formation of bubbles by pipetting slowly.

12. To reduce the settling of organoids onto the bottom of the plate while the Matrigelsolidifies, carefully invert the 96-well plate (surface tension will maintain the Ma-trigel/cell suspension in the well) and place it in a 37◦C, 5% CO2 humidified incubatorfor 30 min.

13. Remove the plate from the incubator and add 75 to 100 μl of modified M87 mediumto each well with a 12-channel pipettor. Incubate the cells overnight at 37◦C in a 5%CO2 humidified incubator.

Treat 3-dimensional cultures with test compoundsPrimary human tumor cells typically proliferate significantly less compared to establishedcell lines. Therefore, longer treatment times with test compounds may be required toassess their effect on proliferation.

14. Dilute test compounds in modified M87 medium to 2× the desired final concentra-tion. Be certain to include proper vehicle (e.g., DMSO) and positive controls (e.g.,cytotoxic compound for viability).

We have successfully used 20 μM doxorubicin or 1 μM staurosporine as a positivecytotoxic control in these assays.

15. Remove the plate from the incubator and add 75 to 100 μl of modified M87 mediumcontaining 2× compound or controls to each well with a 12-channel pipettor to yielda final compound concentration of 1×.

Alternatively, the medium can be removed (discarded) using a 12-channel pipettor bytilting the plate 45◦, making sure not to disturb the Matrigel. Fresh medium containing a1× concentration of the compounds or controls is then added.

16. Incubate the cells for 2 to 5 days at 37◦C in a 5% CO2 humidified incubator.

The majority of primary cells can be cultured without a medium change for severaldays. Since every tumor is different, optimization of medium changes and test compoundconcentrations may be required. Monitor the cultures carefully. Evaporation of the outerwells of a 96-well plate may become an issue when culturing cells for extended periods oftime. When possible, we culture cells in the center wells of the plate and fill unused wellswith sterile water to increase the internal humidity within the plate.

Perform an MTS endpoint viability assay17. Defrost and prepare the MTS reagent according to the manufacturer’s instructions

for the Promega MTS Assay Kit. Transfer the MTS reagent to a 50-ml multi-channelpipetting reservoir.

18. Remove the plate from the incubator and, using a 12-channel pipettor, add 15 to 25 μlof the MTS reagent to each well (10% of the medium volume).

19. Incubate the cells for 1 to 5 hr at 37◦C in a 5% CO2 humidified incubator until thevehicle control wells develop a dark red color.


14.23.30


DMSOcontrol

DMSOcontrol

B

A

800 nMtaxol

0.25 mM 1.0 mM 4.0 mM

valproic acid

16.0 mM 64.0 mM

Figure 14.23.6 Analysis of the viability of patient-derived breast cancer organoids in 3-D culturefollowing 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 ortaxol for 7 days, and imaged using both DIC (bottom rows) and fluorescence microscopy (toprows). Live/dead discrimination was performed using calcein AM (green) to mark viable cells, andethidium bromide homodimer-1 (red) to identify dead cells. Scale bars represents 20 μm. PanelA was adapted from Cohen, et al. (2011) with permission.

20. Remove the plate from the incubator. Using a 12-channel pipettor, transfer 100 μl ofthe medium containing the MTS reagent to a new, clear, flat-bottom 96-well plate.Read the absorbance at 490 nm using a plate reader, and normalize the absorbancevalues to the vehicle controls.

Care should be taken not to disturb the Matrigel and to prevent the formation of bubblesin the culture. Alternatively, the absorbance can be read directly through the Matrigelwithout transferring the medium, but the Matrigel and organoids can cause light scattering,increasing the variability of the results.

Perform live/dead assay21. Thaw live/dead reagents (calcein AM/ethidium bromide homodimer-1) and dilute

them in either DPBS or DMEM/F12 (no serum) to a final concentration of 1 to 5 μM.

22. Remove the plate from the incubator. Using a 12-channel pipettor, carefully aspirateand discard the medium by tilting the plate 45◦, making sure not to disturb theMatrigel.

23. Transfer the diluted live/dead reagents to a 50-ml reservoir. Using a 12-channelpipettor, add 50 to 100 μl of the live/dead reagents to each well. Incubate the cellsfor 15 to 30 min at room temperature.

24. Transfer the plate to a confocal microscope, which can be utilized to capture imagesof calcein AM (ex/em /494/517 nm) fluorescence, which represents the live cells. In


14.23.31


addition, acquire images of ethidium bromide (ex/em /528/617 nm) to identify deadcells.

Subsequently merging the fluorescent images will yield a compiled picture of the relativenumber of live and dead cells in each field of view. Comparison of the relative numbers oflive and dead cells from organoids treated with test compounds and the controls can beused to determine the efficacy of the test compound.

As an example, organoids from a patient were treated with increasing concentrations ofvalproic acid (Fig. 14.23.6A; Cohen et al., 2011). A significant increase in relative numberof dead (red) cells and simultaneous decrease in live cells (green) was observed at higherconcentrations of valproic acid. These results suggest that valproic acid is inducing acytotoxic effect on this patient’s cells. In another example, organoids from a patient weretreated with DMSO or 800 nM Taxol (additional concentrations can also be used). TheTaxol-treated cells had significantly more dead (red) cells compared to the vehicle control,which suggests a cytotoxic effect on these cells. This assay can be performed on numerousprimary patient cells, such as aggregated pleural effusions, to determine if some patientsamples are more resistant to a test compound than others.

SUPPORTPROTOCOL 1

OBTAINING FRESH BREAST TUMOR (e.g., SURGICAL SPECIMENS, BODYFLUIDS, AND METASTASES) FROM PATIENTS

Fresh tumor tissue is used for tissue transplantation and for nucleic acid isolation. Forhigh-quality RNA isolation, it is imperative that the tissue be flash frozen within 10 to 20min after removal from the body to prevent degradation. For tissue transplantation, timeis less critical, but the tissue should remain on ice and in medium until transplantation.We have successfully transplanted tissue after overnight storage on ice.

Materials

Dewar flask of liquid nitrogenRNAlater RNA stabilization reagent (Applied Biosystems, cat. no. AM7021)RPMI 1640 (Fisher Scientific, cat. no. SH30255FS)Bleach70% ethanol

Collection kit: (all reagents and supplies must be sterile)Latex glovesDisposable bibsSterile gauzeSwabsLong, straight trimming bladesSterile scalpelsTissue culture dishesSterile forceps50-ml conical vialsSterile blue, green, and black ink

RNase/DNase-free cryovials50-ml conical tubes (e.g., BD Falcon)

Coordinate tissue collection1. Tissue collection staff checks the surgery schedule daily to identify participants

and assign a “collection event number” (de-identified number) that is not directlyassociated with patient information as required by Health Insurance Portability andAccountability Act (HIPAA) regulations. This ensures that researchers will not beable to identify the patient with this number. The tissue collection staff also reviewsand notes the patient history and the clinical diagnosis.


14.23.32


2. A tissue collection coordinator alerts the surgeon(s), pathologist(s), investigator(s)and tissue repository staff by e-mail 24 hr prior to surgery.

3. Informed consent must be obtained from the patient prior to the tissue collection.This is typically handled by either a nurse or other qualified hospital staff member.

4. Prior to the surgical procedure, the repository technician communicates with theoperating room staff that a specimen for research has been planned.

5. The operating room staff notifies the tissue repository technician 10 min prior to whenthe tissue is removed from the patient. This is to ensure the repository technician canget to the operating room before the tissue is removed from the patient in order tominimize the amount of time to processing (see Tissue Sample Quality Control).

6. When the breast tissue is removed, the technician notes the time of day, and places thetissue into a container on ice for immediate transport to the pathology laboratory. Thespecimen is potentially infectious and must be handled with safety considerationsappropriate for tissue bearing blood-borne pathogens.

7. The pathologist verifies the patient identification, and removes surplus tumor andadjacent uninvolved (normal) tissue for research.

8. Cryovials are labeled with the pre-assigned, de-identified number. The tissue type(cancerous or adjacent uninvolved), location within the breast from where the tissuewas removed, and procurement time are noted on a specimen worksheet.

9. Place three 40-mg pieces from each tissue into separate cryovials, and immediatelyflash freeze in liquid nitrogen (record the time of day). Store the frozen tissues invapor phase liquid nitrogen (−196◦C).

10. Place three additional 20-mg pieces from each tissue type into separate cryovialscontaining 400 μl of RNAlater. Store these tubes at 4◦C overnight. Remove theRNAlater as per manufacturer’s directions and store the specimens at −80◦C.

11. If the remaining tumor specimen is a minimum of 100 mg (300 mg minimum forgrossly uninvolved normal tissue), place the tissues into separate 50-ml conical tubescontaining RPMI for immediate tissue implantation or disassociation to organoids.If the specimens are smaller, the tissue is generally not processed into viable cellsdue to low yields of viable cells. If the minimum amount of tissue is not available forviable cell processing, the remaining tissue is processed as described in steps 9 and10, as necessary.

12. Enter patient identification, specimen collection information, and storage informationinto a biospecimen tracking database such as the itBioPath Biospecimen ManagementSystem (see itBioPath Biospecimen Management System, below).

13. The itBio Path Biospecimen Management system subsequently generates a new de-identified number for each specimen type (RNA, organoids, tissue, etc.) associatedwith that specific patient/collection. The second number is utilized to separately trackeach specimen type. Barcode labels are generated with the de-identified number andare placed on each cryovial. This number is linked in a de-identified manner tosubsequent processing and/or clinical and pathological follow-up information.

itBioPath biospecimen management systemitBioPath is a proprietary system created by the Research Informatics Shared Re-source at Huntsman Cancer Institute that manages, annotates, and tracks storage lo-cations, consent information, protocols, and all associated clinical diagnoses and sur-gical pathology reports. Contact the Research Informatics Shared Resource at HCI(http://www.huntsmancancer.org) for more information about the software suite.


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The itBioPath System complements and enables high quality patient-derived researchwith the following capabilities:

Tracks specimen collections, processing and storage information, banked specimenavailability for originating specimens, succeeding xenograft transformations,and related specimen products.

Provides multigenerational bidirectional linkage of specimen transformations,including the relationship to the originating patient specimen, annotations, andconsents.

Provides separation of patient specimen and xenograft specimen pathologyannotations.

Integrates with genomic experimentation data in the HCI Research Informatics’GNomEx product suite.

Integrates with the University of Utah Health Sciences Electronic Data Warehousefor access to clinical information.

Integrates with a patient-centric system for Cancer Clinical Research (CCR) forcapturing information on patients being treated in the multidisciplinary teams,and others.

Captures chronological and audit documentation of specimen management anddisbursement activities.

Allows for batch data entry capabilities to ensure high quality data collection.Controls security of data access for each user dependent on protocol requirements

and the role of the user interacting with the data.Offers user designed Ad Hoc queries for full data retrieval.

Tissue sample quality controlThe quality of nucleic acid or cell preparations from tissue depends greatly on the qualityof the tissue. In particular, the time required for tissue procurement after removal fromthe patient should be minimized to limit hypoxic effects that can cause loss of tissueintegrity. If the key personnel are aware that collection times are being annotated, thecollection typically proceeds quickly.

14. Always record the procurement time from excision of tissue to freezing or fixation.It is strongly recommended that processing be completed within 20 to 30 min.

15. Before utilizing the RNA, always determine the quality using an instrument such asthe Agilent 2100 Bioanalyzer. Typically, only RNA with an integrity number (RIN)>7 is utilized in downstream applications such as RT-PCR, microarray, etc.

16. Record refrigerator and freezer temperatures daily. Automated alarm systems arerecommended, such as a 24-hr Sensaphone surveillance system.

17. Review and update Standard Operating Procedures at least annually, with input frominvestigators and repository staff.

SUPPORTPROTOCOL 2

PREPARATION OF ESTROGEN PELLETS

Estrogen pellets are made in-house and are a less expensive reliable alternative to pur-chasing them commercially. These pellets are designed to last up to 1 year in mice;however, we typically replace after 6 months.

Materials

Beeswax (Sigma, cat. no. 243221)70% ethanolEstradiol (1,3,5{10}-Estratriene-3, 17 β-diol, Sigma, cat. no. E8875)

22-ml glass sample vial


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Hot plate/magnetic stirrerAluminum foilDry iceWeigh boatsMagnetic stir bar, sterileGlass Pasteur pipetsBunsen burner

1. Weigh 1.95 g of beeswax and place it into a 22-ml glass sample vial.

2. Begin melting on a 60◦C hot plate.

3. Place a sheet of aluminum foil on top of dry ice. Wipe a weigh boat with 70% ethanol,and place it on top of the aluminum foil.

4. As soon as the wax melts, add 50 mg estradiol powder to it. Add a small, sterilemagnetic stir bar to aid the dissolving process.

Typically, it takes 10 to 15 min to dissolve the estrogen at 60◦C.

5. Using a glass Pasteur pipet, drop the wax mixture onto the cooled weigh boat tocreate a pellet consisting of three drops of the melted beeswax solution. Flame thepipet to prevent the wax from solidifying in it.

Each drop will weigh approximately 10 mg, resulting in approximately 30-mg pellets witheach pellet containing ∼1 mg estrogen.

6. Collect the solidified pellets into sterile 1.5-ml microcentrifuge tubes and store up to6 months at 4◦C.

SUPPORTPROTOCOL 3

PREPARATION OF GROWTH FACTOR–REDUCED EHS MATRIX

Growth factor-reduced Matrigel is available from BD Biosciences (cat. no. 54230).Matrigel (EHS matrix) is composed of extracellular matrix derived from Englebreth-Holm-Swarm (EHS) tumors. Large amounts of EHS matrix can be produced in thelaboratory at minimal cost; production of the matrix in the laboratory also allows a singlelot to be used for many experiments, which reduces lot-to-lot variability. The followingprotocol, which is modified from a published procedure (Kibbey, 1994), details a methodto generate EHS matrix. The EHS matrix is obtained by xenografting the EHS cellline (ATCC #CRL-2108) according to the manufacturer’s instructions. Xenografted EHStumors can be flash frozen and stored at −80◦C until needed.

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 rotor250-ml centrifuge bottle (Nalgene, cat. no. 3120-0250)1.5-in. magnetic stir barStir plate (Thermolyne Nuova II)Dialysis tubing (8000 MWCO; Spectrum Labs, cat. no. 132114)


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Snap closures for dialysis tubing1-liter glass beakersDMEM medium with high glucose and L-glutamine (Invitrogen, cat. no.

11965-092)Scissors and hemostat, sterilized15-ml conical tubes, sterile

NOTE: It is important that every step of the protocol be conducted on ice or at 4◦C, orthe solutions will solidify and the entire batch will be unusable.

1. Thaw 10 g of EHS tumor tissue on ice in a 50-ml Nalgene centrifuge bottle (takes∼3 hr).

2. Add 30 ml (3 ml buffer/g tumor) of cold 3.4 M NaCl buffer to the centrifuge tube.Use an electric tissue homogenizer (PowerGen 125) to homogenize the tumor tissueat high speed (setting 6) until the tissue dissolves into a slurry.

3. Place the Nalgene tubes containing the tumor homogenate in a JA-25.50 rotor (Beck-man), balance, and centrifuge 15 min at 8000 × g, 4◦C. Discard the supernatant.

4. Add 30 ml of the 3.4 M NaCl buffer to the tube. Electrically homogenize the tumorpellets again at high speed (setting 6) until the pellet dissolves into a slurry.

5. Balance the tube, and centrifuge 15 min at 8000 × g, 4◦C. Discard the supernatant.

6. Repeat steps 4 and 5.

7. Add 1 ml per g of original tissue of cold 2 M urea buffer to the tube. Electricallyhomogenize the tumor pellets at high speed (setting 6) until the pellet dissolves intoa slurry.

8. Transfer the mixture to a 250-ml Nalgene centrifuge tube. Add a 1.5-in. magnetic stirbar and stir at a medium speed (5 on a scale of 10) overnight at 4◦C on a stir plate.

9. Centrifuge mixture 20 min at 23,708 × g, 4◦C. Save supernatant.

10. Add 0.5 ml per g of original tissue of cold 2 M urea buffer to the tube. Electricallyhomogenize the tumor pellets at high speed until the pellet dissolves into a slurry.

11. Centrifuge the mixture 20 min at 23,708 × g, 4◦C. Save supernatant.

12. Combine supernatants from steps 9 and 11 (14 ml) and place the supernatants indialysis tubing. Seal the dialysis tube with a snap closure.

13. Add at least 10 volumes of cold TBS to a 1-liter glass beaker. Place the dialysis tubingin the beaker with a 1.5-in. magnetic stir bar. Subsequently dialyze by stirring theTBS solution at medium speed (5 out of 10) for 2 hr. Repeat at least two additionaltimes with fresh buffer, for a total of at least 6 hr of dialysis with at least three bufferchanges.

14. Empty the dialysis bags into a 250-ml Nalgene centrifuge bottle with a stir bar andadd 20% (w/v) ammonium sulfate powder (about 16.4g/100 ml, 2.296 g in this case)slowly while stirring at a medium speed (setting 5 on a scale of 10) until all the(NH4)2SO4 is dissolved, then stir at medium speed for 1 hr.

15. Collect the precipitate by centrifugation for 15 min at 7000 × g, 4◦C. Discard thesupernatant.

16. Add 14 ml of cold TBS and homogenize the pellet at high speed (setting 6) until thepellet breaks up into a slurry. Add a stir bar and stir at medium speed (5 on a scale of10) until the slurry dissolves.


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17. Transfer the solution to dialysis tubing and seal the tube with a snap closure. Add atleast 10 volumes of cold TBS to a 1-liter glass beaker. Place the dialysis tubing inthe beaker with a 1.5-in. stir bar. Subsequently dialyze by stirring the TBS solutionat medium speed (5 on a scale of 10) for 2 hr. Repeat the dialysis with fresh TBS forat least 2 more hr.

18. Repeat steps 14 and 15.

19. Add 7 ml of cold TBS and homogenize the pellet at high speed (setting 6) until thepellet breaks up into a slurry. Add a stir bar and stir at medium speed (5 on a scale of10) until the slurry dissolves.

20. Transfer the solution to dialysis tubing and seal the tube with a snap closure. Add atleast 10 volumes of cold TBS containing 5 ml chloroform per liter to a 1-liter glassbeaker. Place the dialysis tubing in the beaker with a 1.5-in. stir bar. Subsequentlydialyze by stirring the TBS/chloroform solution at medium speed (5 on a scale of 10)until the mixture becomes clear, which typically can take between 2 and 10 hr.

A glass container or cylinder must be used since the solution contains chloroform, whichis utilized to sterilize the mixture.

21. After the mixture is no longer cloudy, remove the dialysis bag, clean the containerthoroughly to remove the chloroform, and continually dialyze for at least 2 hr againstat least 10 vol of cold TBS with stirring at a medium speed (5 out of 10). Repeatthe dialysis at least three more times with fresh buffer, for a total of at least 8 hr ofdialysis with at least four buffer changes.

22. Perform the final dialysis against at least 10 vol of DMEM medium in a 1-liter glassbeaker with medium stirring (5 on a scale of 10) overnight at 4◦C.

23. In a biosafety cabinet, spray the outside of the dialysis bag with 70% ethanol thenopen and hold the bag with sterile scissors and hemostats.

24. Remove the EHS matrix with a sterile pipet and/or pour it into a sterile 15-ml conicaltube on ice. Aliquot the matrix into sterile 1.5-ml microcentrifuge tubes on ice.

This protocol typically generates a final EHS matrix protein concentration of 6 to 7 mg/ml.The aliquots can be stored at 4◦C for up to 1 month or at −20◦C for up to 1 year. Makesure to thaw frozen aliquots at 4◦C.

REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Collagenase enzyme stock (10×)

For human tumors: Prepare 20 mg/ml Type 3 collagenase (Worthington, cat. no.LS004182) in Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen, cat. no.14040-141). Filter sterilize and store in aliquots at −20◦C for up to 1 year.

For xenografted or murine tumors: Prepare 10 mg/ml Type IV collagenase (Sigma,cat. no. C5138) in digestion buffer (for xenografted or murine tumors; see recipe).Filter sterilize and store in aliquots at −20◦C for up to 1 year.

Digestion buffer

For human tumors:DMEM-F/12 (1:1) with HEPES (Thermo Scientific, cat. no. SH30023.01) supple-

mented with:continued


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10 mM HEPES (Sigma, cat. no. H3537)2% bovine serum albumin (BSA, Sigma, cat. no. A7906)5 μg/ml insulin (with transferrin/selenium, Invitrogen, cat. no. 51500-056)0.5 μg/ml hydrocortisone (Sigma, cat. no. H0888)50 μg/ml gentamycin (HyClone, cat. no. 3V30080.01)Sterile filter and store up to 1 month at 4◦C Use within 1 month.

For xenografted or murine tumors:Hyclone RPMI 1640 (Thermo Scientific, cat. no. SH30255.01) supplemented with:2.5% (v/v) FBS10 mM HEPES (Sigma, cat. no. H3537)1× pen-strep (HyClone, cat. no. SV30010)Filter sterilizeStore up to 30 days at 4◦C

HBEC freezing medium

HBEC medium (see recipe) supplemented with:5% (v/v) fetal bovine serum (FBS; Sigma, cat. no. F6178)10% (v/v) dimethylsulfoxide (DMSO; Sigma, cat. no. D2650)Filter sterilizeStore up to 30 days at 4◦C

Do not confuse with tissue freezing medium (see recipe).

Human breast epithelial cell (HBEC) medium

Hyclone DMEM/F12 (1:1) with HEPES (Thermo Scientific, cat. no. SH30023.01)supplemented with:

10 mM HEPES (Sigma, cat. no. H3537)5% (v/v) fetal bovine serum (FBS, Sigma, cat. no. F6178)1 mg/ml bovine serum albumin (BSA, Sigma, cat. no. A7906)1 μg/ml insulin (with transferrin/selenium, Invitrogen, cat. no. 51500-056)0.5 μg/ml hydrocortisone (Sigma, cat. no. H0888)50 μg/ml gentamycin (HyClone, cat. no. 3V30080.01)2.5 μg/ml Fungizone (Amphotericin B, HyClone, cat. no. SV30078.01)Filter sterilizeStore up to 30 days at 4◦C

Hyaluronidase enzyme stock (100×)

Prepare hyaluronidase (Sigma, cat. no. H3884) at 10,000 U/ml in Dulbecco’sphosphate-buffered saline (DPBS; Invitrogen, cat. no. 14040-141). Filter sterilizeand store at −20◦C for up to 1 year.

Lidocaine-bupivacaine mixture

5 mg/ml Lidocaine (Hospira, cat. no. 0409-2066-05; http://www.hospira.com)2.5 mg/ml Bupivacaine (available at pharmacies)Dilute in HBSS (Thermo Scientific, cat. no. SH30268.02)Filter sterilize, aliquot in 1.5-ml microcentrifuge tubes and store at room tempera-

ture until the expiration date listed on the lidocaine and bupivacaine samples

Modified M87 medium

Prepare fresh stocks of the different medium components described in Table 14.23.4using sterile technique. The stock solutions can be stored for up to 1 month at−20◦C, except cholera toxin and T3, which are stored at 4◦C.

continued


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Hyclone DMEM/F12 (1:1) (Thermo Scientific, cat. no. SH30023.01)Hyclone characterized fetal bovine serum (Thermo Scientific, cat. no. SH30071.03)Insulin-transferrin-selenium-X supplement (100×) (Invitrogen, cat. no. 51500-

056)Penicillin-streptomycin-glutamine (100×), liquid (Invitrogen, cat. no. 10378-016)Human recombinant epidermal growth factor (EGF) (BD Biosciences, cat. no.

354052)Hydrocortisone (Sigma, cat. no. H0135)Cholera toxin (Sigma, cat. no. C8052)3,3′,5-triiodo-L-thyronine (T3) (Sigma, cat. no. T0281)β-estradiol (E2) (Sigma, cat. no. E2758)(±)-isoproterenol hydrochloride (Sigma, cat. no. I6504)Ethanolamine (Sigma, cat. no. E9508)O-phosphorylethanolamine (Sigma, cat. no. P0503)Add the specified volume of medium component stocks to DMEM/F12 to achieve

the appropriate final concentration of each supplement as instructed in Table14.23.4. Pass the modified M87 medium through a 0.22-μm filter.

The medium can be stored up to 1 week at 4◦C.

NaCl buffer (3.4 M), pH 7.4

397 g NaCl25 ml 2 M Tris buffer, pH 7.4 (see recipe)1.8 liter deionized water3.0 g EDTA (ethylenediaminetetraacetic acid) (Sigma, cat. no. E9884)0.5 g NEM (N-ethylmaleimide; Sigma, cat. no. 692905)Adjust pH to 7.4Store at room temperature for up to 6 months

TAC solution (1×)

Mix 1 part of 170 mM Tris buffer, pH 7.4 (see recipe) with 9 parts of 150 mMNH4Cl, pH 7.4. Filter sterilize and store at room temperature for up to 6 months.

Tissue freezing medium

95% (v/v) FBS5% (v/v) DMSOFilter sterilizeStore up to 1 month at 4◦CDo not confuse with HBEC freezing medium.

2 M Tris buffer, pH 7.4

242.28 g Tris base800 ml deionized waterAdjust pH to 7.4 with HClAdd deionized water to 1 literStore at room temperature for up to 6 months

Tris-buffered saline (TBS)

12.1 g Tris base18 g NaCl1.8 liter deionized waterAdjust pH to 7.4 with HClAdd deionized water to 2 litersFilter sterilizeStore at room temperature for up to 6 months


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Table 14.23.4 Preparation of 500 ml of Modified M87 Medium for Culturing Primary orXenografted Human Tumor Cells

ComponentStockconcentration

Finalconcentration

Volume of stock for500 ml of medium

DMEM/F12 479 ml

Fetal bovine serum (FBS) 2% 10 ml

Insulin-transferrin-selenium-X 100× 1× 5 ml

Penicillin-streptomycin-glutamine 100× 1× 5 ml

Human epidermal growth factora 100 μg/ml 5 ng/ml 25 μl

Hydrocortisone b 10 mg/ml 0.3 μg/ml 15 μl

Cholera toxinc 10 μg/ml 0.5 ng/ml 25 μl

3,3′,5-Triiodo-L-thyronine (T3)d 200 μM 5 nM 12.5 μl

β-Estradiol (E2)b 20 μM 0.5 nM 12.5 μl

(±)-Isoproterenol hydrochloridee 5 mM 5 μM 500 μl

Ethanolaminec,f 100 mM 50 nM 250 μl

O-Phosphorylethanolaminec,f 100 mM 50 nM 250 μlaStock prepared in DPBS.bStock prepared in absolute ethanol.cStock prepared in deionized water.dT3 was dissolved in deionized water and a drop of 0.1 M NaOH was added and was passed through a 0.22-μm filter.eStock was prepared in 95% ethanol in a fume hood.fSolution was passed through a 0.22-μm filter.

Urea buffer (2 M), pH 7.4

240 g urea12.1 g Tris base18 g NaCl1.8 liter deionized waterAdjust pH to 7.4 with HClAdd deionized water to 2 litersStore at room temperature for up to 2 months

COMMENTARY

Background InformationPatient-derived tumorgrafts growing in

mice can recapitulate clinical manifestationsand pathological features of a patient’s orig-inal tumor, including its histology, molecularsubtype, genomic organization, and metastaticdissemination (DeRose et al., 2011). Thus,mice carrying primary tumorgrafts are idealexperimental models for studying the in vivoevents of human tumorigenesis and for as-sessing the efficacy of investigational com-pounds in diverse cancer subtypes. Tumor-grafting also provides a method to propagatepatient-derived tissue for more extensive invivo and in vitro studies. While the compli-cated methodology, cost, variability, and timerequirements of tumorgrafting make this tech-

nique impractical for widespread adoption intothe clinic, there are potential opportunities toapply tumorgrafts in future clinical practice.For example, tumorgrafts can provide diag-nostic information on the aggressiveness of apatient’s tumor [successful engraftment is as-sociated with worse patient outcome (DeRoseet al., 2011)], and for functionally evaluatingthe sensitivity of tumors to specific drugs priorto treating patients. While tumorgrafts are get-ting traction in early translational research pro-grams, significant barriers must still be over-come before they are practical for clinical ap-plications.

A tumorgraft bank is an effective tool formany research purposes. Currently, the rate-limiting step is the initial investment, in both


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cost and time, to establish a tumorgraft bankthat not only spans the breadth of cancer sub-types but also contains replicates of each tu-mor subtype. Once a diverse bank of trans-plantable tissue is categorized into molecu-lar and genomic subtypes, incorporation ofpatient-derived tissue into basic research anddrug discovery platforms becomes signifi-cantly more practical. Ideally, a centralizedrepository would be established to consolidateand disseminate tissue samples, which wouldeliminate the most significant barriers asso-ciated with generating individual banks andincrease the diversity of tumor subtypes avail-able for research. Until then, the protocols de-scribed in this unit should facilitate in-housedevelopment of tumorgraft banks.

One of the most suitable applications fortumorgrafts in drug discovery is preclinicalevaluation of select compounds in a “pseudo-clinical study” or “Phase 2 mouse study.” Newchemical entities in early development can betested against cohorts of tumors representingspecific molecular subtypes to determine dif-ferential activity across a broad spectrum oftumors. An understanding of a compound’srange of activity in diverse tumor subtypes hasbecome increasingly important when design-ing clinical trials, particularly with targetedtherapies that are often effective only in tumorswith specific oncogenic drivers. Thus, tumor-grafts can fill a critical void between conven-tional preclinical cancer models and clinicaltrials in patients.

While tumorgrafts provide distinct advan-tages over cell lines or xenografted cell lines,they are still not ideal for all basic researchpurposes due to their many limitations. Thecritical hurdles of generating tumorgrafts in-clude regulatory processes, such as Institu-tional Review Board and HIPPA approvals;establishing the infrastructure and collabora-tions necessary to obtain surgical specimensfrom patients; cost to obtain, cryopreserve,transplant, and characterize specimens; longtumor propagation times in mice; necessityfor growth in immune-compromised hosts;and challenges with performing in vitro ex-perimental manipulations. Thus, tumorgraftsare best employed in focused studies to moreclosely establish relevance of an experimentalobservation to the human disease. Similarly,tumorgrafts would not be efficient in high-throughput drug screening; rather, they wouldbe more effectively employed to validate spe-cific lead compounds as a step toward bringinga drug to the clinic.

Critical ParametersWhen establishing a tumorgraft bank, the

most critical parameter is the quality of the sur-gical specimen. Clearly, tumor samples withlow viability are inadequate for tumor graft-ing. Thus, a reliable research team with de-fined roles in obtaining and processing surgi-cal specimens is absolutely critical. All post-collection procedures described in this unitcan be optimized and customized as neces-sary for experiments, but these efforts are invain without quality specimens. To begin, aninvestigator should establish a repertoire withall members of the clinical team involved incollecting the tissue, including administrativestaff, nurses, surgeons, pathologists, and re-searchers. The team must collect and distributethe surgical specimens to laboratory person-nel within a defined time, and understand thattissue quality is affected by several variablesassociated with collection and processing,such as time-to-transplant and sterility of thesample. Quarterly meetings with the entireteam for general discussions and presentationson the quality of tissue specimens, updates onresearch studies utilizing tissue sample, andreviewing banking procedures is one effec-tive method to keep the team informed andmotivated. It is also important to recognizethe efforts of everyone involved by includ-ing individuals critical to the collections onmanuscripts, including them as key person-nel on grant applications, and acknowledgingthem in presentations.

Once a tissue bank is generated, it is im-portant to assess the molecular and histologicaldiversity of the samples and to compare the tu-morgrafts with the originating tumor. For this,it is important to obtain de-identified medi-cal reports that provide diagnoses, treatments,medications, tumor pathology, and if possible,paraffin blocks of the patient’s tumor. Molec-ular and histological analyses, as described inDeRose et al. (2011), can then be performedto ensure that the tumor graft faithfully repli-cates the patient’s tumor. While in the major-ity of cases a tumorgraft closely replicates thepatient’s tumor, we have observed one casein which a human lymphoma arose follow-ing transplantation of a breast cancer surgi-cal specimen from an 80-year old individual.Thus, tumorgrafts should be carefully moni-tored for their ability to replicate the molecu-lar, genomic, and histologic phenotypes of thepatient’s primary tumor.

Each patient-derived tumor sample isunique. As such, the method used for


14.23.41


processing tissues is dynamic and requirescareful monitoring and alterations as needed.While this unit provides a standard protocolthat works well for many tumorgrafts, modi-fications to the method may be necessary tooptimize processing of different samples. Assuch, the educated judgment of an experiencedresearcher is often required. For example, theabundance and composition of stroma and ex-tracellular matrix may differ between tumortissues, which directly affects the time neededfor enzymatic digestion by collagenase andhyaluronidase. Other variables include the sizeof the surgical specimen, level of necrosis,quantity of red blood cells and inflammatorycells, and contamination with bacteria or yeast,all of which affect tissue processing and futureapplications. During the procedure, the tissuesample should be carefully monitored bothvisually and microscopically for the degreeof enzymatic digestion, cellular morphology(signs of necrosis/apoptosis), and viscosity ofthe medium (resulting from release of DNAfrom dead cells) to assess the quality of thepreparation. In addition, enrichment of tumororganoids from debris, stroma, and inflamma-tory cells should be monitored microscopi-cally, with adjustments made to differentialcentrifugation steps as described in the unit.These observations, and any other informationabout the quality or viability of the preparation,should be noted for each sample and uploadedto the tumor bank’s database. Careful observa-tion and procedural adjustments during tissueprocessing should improve the quality of thepreparation.

TroubleshootingLack of growth: Once a transplantable tu-

mor graft line has been established, the suc-cessful re-transplantation of the line is high(>90%). The most common cause for lack ofgrowth of an established tumor graft is thatthe tumor fragment that was implanted into thehost was of low quality (e.g., containing morestroma than tumor or extensive necrosis) or be-cause of a failure to supplement mice carryingER+ tumors with estrogen. If necrosis lim-its the growth of tumorgrafts, co-engraftmentof human mesenchymal stem cells can stimu-late tumor angiogenesis and promote growth(DeRose et al., 2011). If primary, patient-derived tumor tissue fails to engraft upon firstimplantation, it may be among the 70% to 74%of breast tumors that do not engraft using themethods in this unit. It is more efficient tofirst establish a bank of established tumorgraft

lines than rely on primary surgical specimensfor experiments.

Genetic or phenotypic drift: Serially trans-planted tumorgrafts sometimes tend to growfaster than earlier passages (DeRose et al.,2011). Careful observation of the tumor char-acteristics and behavior is important, as “drift”can be a problem. To retain the featuresof the original tumor as much as possible,we strongly suggest maintaining many frozenvials of early passage tumorgraft fragments forfuture implantation.

Anticipated ResultsUsing these protocols, the origin of the

breast tumor (primary or metastatic site),ER/PR status, or HER2 status did not re-liably predict successful engraftment of theoriginal tumors into mice in our study us-ing approximately 50 patient samples (DeRoseet al., 2011). However, positive engraftmentcorrelated with poor patient outcome, indepen-dently of the aforementioned factors (DeRoseet al., 2011). In patients, triple-negative tu-mors (those testing negative for estrogen re-ceptor, progesterone receptor, and HER2) aretypically aggressive. Correspondingly, oncethey are engrafted, they tend to grow fasterthan other subtypes of breast cancer (DeRoseet al., 2011). Primary tumor tissues from pa-tients who have been treated with neoadju-vant chemotherapy have not successfully en-grafted, at least in our experience thus far.By contrast, metastases do engraft even afterchemotherapy or radiation therapy. We gen-erally allow 6 months after the first engraft-ment for a tumor to grow. If growth is notdetected by 6 months, the engraftment is con-sidered unsuccessful. Based on attempted en-graftment of approximately 50 different pa-tient tumors into NOD/SCID mice, the en-graftment rate from surgical specimens was30% to 36% (DeRose et al., 2011). Althoughthe overall engraftment rate for surgical speci-mens was relatively low, when one tissue frag-ment from a patient grows, all transplantedfragments from that patient generally grow.Likewise, when a tumor does not successfullyengraft, none of the fragments grow. Oncea tumor has successfully engrafted, approxi-mately 100% of subsequent transplants fromthat line can be expected upon serial passagewithout intervening culture steps. It is impor-tant to note that engraftment data discussed inthis unit were obtained from tissue/cells thatwere not cultured, and it is unknown whetherculturing, in either monolayer or 3-D, will


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have an effect on the phenotype of subsequenttumorgrafts.

Time ConsiderationsGrowth rates of patient-derived tumor sam-

ples in mice are highly variable. To date,our fastest-growing tumorgraft became pal-pable around 2 weeks after transplantation,and was harvested approximately 2 monthsfollowing engraftment. It is common for tu-mor grafts to take several months to becomepalpable and 7 to 8 months to be ready toharvest for the first time. Careful palpationis critical to avoid termination of tumorgraftlines that otherwise might be successful. Whenre-transplanting characterized tissue, an ad-ditional month should be added to the ex-pected growth time if the tumor fragment isfrozen prior to implantation. Often, the re-transplanted tissue will have reduced latencycompared to the original patient-derived sam-ple. When cohorts of mice are transplantedwith similar size fragments or cell numbersfrom the same tumorgraft sample, tumors typ-ically grow with similar latency.

AcknowledgementsThe work described in this article was

funded by grants from the National Institutesof Health (CA143815 and CA140296), the De-partment of Defense (W81XWH-09-1-0431and W81XWH-08-1-0109), and the Ameri-can Association for Cancer Research with theBreast Cancer Research Foundation (07-60-26-WELM), as well as with funds from theHuntsman Cancer Foundation/Institute.

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Morrison, S.J., and Clarke, M.F. 2003. Prospec-tive identification of tumorigenic breast cancercells. Proc. Natl. Acad. Sci. U.S.A. 100:3983-3988.

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