Trafficking of Metastatic Breast Cancer Cells in Bone PRINCIPAL INVESTIGATOR:

Purpose: In vivo studies have focused on the latter stages of the bone metastatic process(osteolysis), whereas little is known about earlier events, e.g., arrival, localization, and initialcolonization. Defining these initial steps may potentially identify the critical points susceptible totherapeutic intervention.Experimental Design: MDA-MB-435 human breast cancer cells engineered with greenfluorescent protein were injected into the cardiac left ventricle of athymic mice. Femurs wereanalyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry,and histomorphometry at times ranging from1hour to 6 weeks.Results: Single cells were found in distal metaphyses at 1hour postinjection and remained assingle cells up to 72 hours. Diaphyseal arrest occurred rarely and few cells remained there after24 hours. At1week, numerous foci (2-10 cells)were observed, mostly adjacent to osteoblast-likecells. By 2weeks, fewer but larger foci (z50 cells)were seen.Most bones hada single largemassat 4weeks (originating froma colonyor coalescing foci)which extended into the diaphysis by 4 to6weeks. Little change(<20%) inosteoblast or osteoclast numbers was observed at 2weeks, butat 4 to 6weeks, osteoblastswere dramatically reduced (8%of control), whereas osteoclastswerereducedmodestly (tof60% of control).Conclusions: Early arrest in metaphysis and minimal retention in diaphysis highlight the impor-tanceof the localmilieu indeterminingmetastaticpotential.These results extend theSeedandSoilhypothesis by demonstrating both intertissue and intratissue differences governing metastaticlocation. Ours is the first in vivo evidence that tumor cells influencenot only osteoclasts, aswidelybelieved, but also eliminate functional osteoblasts, thereby restructuring the bone microenviron-ment to favor osteolysis.The datamay also explainwhy patients receiving bisphosphonates fail toheal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblastfunction are needed to effectively treat osteolytic bonemetastases. Breast cancer has a remarkable predilection to colonize bone,with an incidence between 70% and 85% in patients (1–3). Atthe time of death, metastatic bone disease accounts for the bulkof tumor burden (4). For women with bone metastases, thecomplications—severe, often intractable pain, pathologic frac-tures, and hypercalcemia—are catastrophic. Despite its obviousclinical importance, very little is understood about the funda-mental mechanisms responsible for breast cancer metastasis tobone. Research progress has been hampered by the dearth of,and technical difficulties inherent in, the current models.Most models of metastasis poorly recapitulate the pathogen-esis of breast cancer. The ideal model would involve dis-semination from an orthotopic site (i.e., mammary fat pad),colonization, and osteolysis. None of the currently availablehuman breast xenograft models spread to bone following ortho-topic implantation and only one murine model metastasizes tobone from the mammary fat pad (5). Furthermore, most humancell lines do not metastasize to bone in mice regardless of routeof injection. The most commonly used model of breast cancermetastasis to bone involves injection of tumor cells into thearterial circulation via the left ventricle of the heart (4, 6–8). Thisroute of injection minimizes first-pass filtration through pulmo-nary capillaries, thereby allowing more cells to reach the bone.Human Cancer Biology Authors’Affiliations: Departments of Pathology and Medicine-Hematology/Oncology, Comprehensive Cancer Center, Center for Metabolic Bone Disease,National Foundation for Cancer Research, Center for Metastasis Research,University of Alabama at Birmingham, Birmingham, Alabama, andDepartment ofBiochemistry and Molecular Biology, Pennsylvania State University, UniversityPark, PennsylvaniaReceived 8/19/05; revised11/1/05; accepted12/15/05.Grant support: U.S. Army Medical Research and Materiel Command (DAMD-17-02-1-0541, DAMD-17-03-01-0584, and DAMD17-02-1-0358) and the Universityof Alabama at Birmingham Breast Specialized Programs of Research Excellence(P50-CA89019). Additional support was provided by CA87728, the NationalFoundation for Cancer Research, Center for Metastasis Research, and thePennsylvania Department of Health Breast Cancer Program.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: P.A. Phadke and R.R. Mercer contributed equally to this work.This work was submitted in partial fulfillment of the requirements for the Universityof Alabama at Birmingham Graduate Program in Molecular and Cellular Pathology(P.A. Phadke) and Penn State Graduate Program in Biochemistry and MolecularBiology (R.R. Mercer).Requests for reprints: Danny R.Welch, Department of Pathology, University ofAlabama at Birmingham, Volker Hall G-019A, 1670 University Boulevard,Birmingham, AL 35294-0019. Phone: 205-934-2961; Fax: 205-975-1126;E-mail: [email protected] American Association for Cancer Research.doi:10.1158/1078-0432.CCR-05-1806 www.aacrjournals.orgClin Cancer Res 2006;12(5) March1, 20061431 Current methods to detect bone metastases are insufficientlysensitive (e.g., radiography) or are impractical for adequatelystatistically powered experiments because of costs or labor-intensiveness. Radiography can detect osteolytic lesions onlyafter more than half of the calcified bone matrix has beendegraded (9). Microcomputerized tomography is not widelyavailable, but is likewise of insufficient resolution to recognizesingle tumor cells. Serial sectioning (which would be requiredto locate rare single cells) is cost-prohibitive, except for smallstudies. As a result, experiments have been limited to late eventsof metastatic bone disease, such as osteolysis. Therefore,antecedent events (i.e., arrival, lodging, intraosseous trafficking,and colonization) have not been studied except by inference.To overcome some of the technical limitations, moresensitive methods using reporter molecules, such as luciferase(10) or h-galactosidase (LacZ) have recently been described(11–13). Luciferase, although it allows for in situ detection oftumor cells in the bone, does not allow for microscopiclocalization of the cells. Because luminescence depends ona fully viable cell, use of luciferase is limited ex vivo . h-Galactosidase is excellent for studies at the histologic level butcannot be used for studies involving intact bone unless thelesions are macroscopic. Diffusion or distribution of substrateinto bone is also a complication.Fluorescent molecules, like enhanced green fluorescentprotein (GFP), have also been employed with some success inthe early detection of bone metastasis (14–16). We recentlyused the GFP-tagged MDA-MB-435 metastatic human breastcancer cell line to reveal formation of osteolytic bone lesionsfollowing intracardiac injection in athymic mice (15). Likeluciferase, GFP can be used to detect lesions in situ , even thoughthe limits of detection are restrictive (f0.5-1 mm). Duringexperiments designed for other purposes, we detected singletumor cells in bone within minutes postinjection. Because tothe best of our knowledge, no one had ever systematicallystudied the earliest tumor cell-bone interactions (except byserendipitous histologic sections), we decided to use the powerof GFP to begin addressing the early events associated withbreast tumor cells that have already disseminated to bone.It has long been recognized that, once cells arrive in thebone, they alter homeostasis. Turnover of the skeleton isdynamic and continuous throughout embryonic developmentand adulthood. Calcified bone matrix turns over completely,on average, every decade (17, 18). Calcified matrix remodelinginvolves an interplay between osteoblasts (bone forming cells)and osteoclasts (bone resorbing cells). Altering the balance ofactivities results either in excessive bone deposition (osteopet-rosis) or bone loss (osteoporosis). Although larger individualbone lesions contain regions that are both osteopetrotic andosteoporotic, most breast cancer bone metastases are notosteolytic. The current paradigm suggests that tumor cellsinfluence osteoclast activity (4, 19). Using the GFP model ofbreast cancer metastasis to bone, we sought to identify keytumor cell–bone cell interactions (and the timing of thoseinteractions) that occur during the pathogenesis of bonemetastasis. Materials andMethods Cell lines and culture. Metastatic human breast carcinoma cell line,MDA-MB-435 (MDA-435), a generous gift from Dr. Janet Price(University of Texas M.D. Anderson Cancer Center, Houston, TX),was stably transfected with pEGFP-N1 (BD Biosciences Clontech, PaloAlto, CA) by electroporation (Bio-Rad Model GenePulser, Hercules, CA;220 V, 960 A Fd, 1V) or transduced with a HIV type 1-based, lentiviralvector system constitutively expressing enhanced GFP (20, 21). For thelentivirus, the GFP coding sequence was inserted into the vector 5V ofthe internal ribosome entry site and puromycin sequences, each ofwhich were under control of the early cytomegalovirus promoter.Infectious stock were prepared by transfection of 293T cells and used ata multiplicity of infection of f10.The origin of MDA-MB-435 has been questioned because the cellsexpress melanoma-associated genes in cDNA microarray experiments.However, the patient was reported only to have a breast carcinoma.Because MDA-MB-435 cells express milk proteins (22), it is mostsimple to conclude that the cells are poorly differentiated breastcarcinoma.Parental cells were cultured in a mixture (1:1 vol/vol) of DMEM andHam’s F12 media (DMEM/F12; Invitrogen, Carlsbad, CA) supple-mented with 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 0.02mmol/L nonessential amino acids, 5% fetal bovine serum (AtlantaBiologicals, Norcross, GA), without antibiotics or antimycotics (cDME/F12). All cultures were confirmed to be negative for Mycoplasma spp.infection using a PCR-based test (TaKaRa, Shiga, Japan).GFP-expressing cells were grown in cDME/F12 plus G418 (Geneticin,500 Ag/mL, Invitrogen) or puromycin (500 Ag/mL, Fisher Scientific,Hampton, NH). The brightest 15% (lentiviral) or 25% (pEGFP)fluorescing cells were sorted using either Coulter EPICS V cell sorter(Beckman-Coulter, Fullerton, CA) or a BD FACSaria cell sorter (BDBiosciences Immunocytometry Systems, San Jose, CA).Intracardiac injections. Cells at 80% to 90% confluency weredetached using a mixture of 0.5 mmol/L EDTA and 0.05% trypsin inCa-, Mg-, and NaHCO3-free HBSS. Viable cells were counted using ahemacytometer and resuspended at a final concentration of 1.5 10cells/mL in ice-cold HBSS. Cells were not used unless viability was>95%, but was usually >98%. Female athymic mice ages between 4 and6 weeks (Harlan Sprague-Dawley, Indianapolis, IN) were anesthetizedby i.m. administration of a mixture of ketamine-HCl (129 mg/kg), andxylazine (4 mg/kg). Cells (3 10 in 0.2 mL) were injected into the leftventricle of the heart between the third and fourth or between thefourth and fifth intracostal space. The presence of bright red, asopposed to burgundy, colored blood prior to and at the end of eachinoculation confirmed injection of the entire volume into the arterialsystem. Mice were necropsied at 1, 2, 4, 8, 24, 48, and 72 hours and 1,2, 4, and 6 weeks postinoculation following anesthesia with ketamine/xylazine and euthanasia by cervical dislocation. At least two indepen-dent experiments were done with 5 to 12 mice per experimental group.Not all time points were collected for every experiment.Although widespread skeletal metastases develop after intracardiacinjection (15, 23), the experiments reported here focused exclusively onthe femur, a common site for metastasis that is easily accessible. Thefemurs were removed and examined by low magnification ( 2-10)fluorescence stereomicroscopy and histologic and histomorphometricanalyses (24, 25). Some femurs were divided into proximal and distalmetaphyses plus cortical shaft (diaphysis) from which the marrow wascollected and cells examined by flow cytometry or quantitative real-timePCR. Corroborating experiments were done with the contralateralfemur to assure that there was no bias for sidedness.Mice were maintained under the guidelines of the NIH, theUniversity of Alabama at Birmingham, and the Pennsylvania StateUniversity. All protocols were approved and monitored by theappropriate Institutional Animal Care and Use Committees.Fluorescence microscopy. To visualize metastases derived from theGFP-tagged cell lines, whole femurs (dissected free of soft tissue using ano. 11 scalpel blade with gauze used to grip and remove tissueremnants) were placed into Petri dishes containing ice-coldCaandMg-free Dulbecco’s PBS and examined by fluorescence microscopyusing a Leica MZFLIII dissecting microscope with 0.5 objective andHuman Cancer Biology www.aacrjournals.orgClin Cancer Res 2006;12(5) March1, 20061432 GFP fluorescence filters(kexcitation = 480 F 20 nm;kemission, 510 nmbarrier; Leica, Deerfield, IL). Photomicrographs were collected using aMagnaFire digital camera (Optronics, Goleta, CA), and ImagePro Plus5.1 software (Media Cybernetics, Silver Spring, MD).Bone fixation and storage. Intact, dissected femurs from individualmice were placed in 25 mL glass scintillation vials and fixed in freshlyprepared 4% paraformaldehyde inCaand Mg-free Dulbecco’s PBSor in periodate-lysine-paraformaldehyde solution (26) at 4jC for 24 to48 hours. GFP fluorescence was difficult to maintain in fixed tissues andbone sections; however, we were able to overcome this limitation bymaintaining the samples at 4jC (27). Bones destined for histologicsectioning were subsequently removed and decalcified in 0.5 mol/LEDTA in Caand Mg-free Dulbecco’s PBS.Bone histomorphometry. Bones were dehydrated in increasingconcentrations of ethanol and embedded in a mixture of 80:20 methylmethacrylate and dibutylphthalate. Serial coronal sections (5 Am) wereobtained using a Leica 2265 microtome. The distal ends of femurs(spongiosa) were analyzed. Sections were first stained with Sanderson’srapid bone stain for 2 minutes. Once tumor cells were identified,subsequent sections were stained with Goldner’s trichrome and tartrate-resistant acid phosphatase (TRAP). Histomorphometry was done at theUniversity of Alabama at Birmingham Center for Metabolic BoneDisease Histomorphometry and Molecular Analysis Core Facility by themethod of Parfitt et al. (24, 25) using Bioquant image analysis software(R&M; Biometrics, Nashville, TN).Immunohistochemistry. Paraffin-embedded samples were sectioned(5 Am, coronal or sagittal), deparaffinized, and rehydrated beforeantigen retrieval by microwaving for f8 minutes at full power (700 W)in a 10 mmol/L citrate buffer (pH 6). Samples were boiled for 5minutes in the microwave oven. Endogenous peroxidase activity wasblocked by treatment with 3% hydrogen peroxide for 5 minutes.Sections were blocked with 1% goat serum for 1 hour. Slides wereincubated with primary rabbit polyclonal anti-GFP IgG (1:250;Molecular Probes, Eugene, OR) for 1 hour, followed by secondarybiotinylated anti-rabbit antibody (TITRE; Level 2 Ultra StreptavidinDetection System, Signet Labs, Dedham, MA). Detection was achievedusing Biogenex liquid DAB kit (Biogenex, San Ramon, CA) and slideswere counterstained using hematoxylin. GFP-positive tumor samplesserved as positive controls. Negative controls were done by omitting theprimary antibody.Some femurs were fixed in a solution of 2% paraformaldehydecontaining 0.075 mol/L lysine and 0.01 mol/L sodium periodate (pH7.4), 4jC for 24 hours in an attempt to maintain alkaline phosphataseactivity (26). Although the alkaline phosphatase activity was not wellpreserved, fluorescence was maintained; fluorescent MDA-435 cellscould be observed in femurs taken throughout the time course.Following decalcification as described above, the bones were embeddedin paraffin. Paraffin-embedded bones were sectioned lengthwise into 10Am sections and several sections throughout the bone were analyzed.The sections were deparaffinized, rehydrated, and stained for apoptoticcells with a modified terminal nucleotidyl transferase–mediated nickend labeling (TUNEL) procedure using Cy-5 rather than FITC-labeleddUTP (28). Bone sections were first scanned at 20 magnification usinga fluorescence confocal microscope. Areas in which GFP-positive cellswere detected were further analyzed at 40 magnification with bothfluorescence and phase microscopy. Fluorescent images were capturedat two wavelengthskexcitation = 480 F 20 nm(kemission, 520 nm for GFP)andkexcitation = 647 nm(kemission, 670 nm for Cy-5). A comparison ofthe numbers of breast cancer cells detected by fluorescence microscopyversus use of anti-GFP gave essentially the same trends (data notshown).TRAP-positive cells were determined in the femurs of mice at varioustimes following inoculation with metastatic breast cancer cells. Two toeight sections from two to four bones per time period were stained forthe presence of TRAP by immunohistochemistry (Sigma-Aldrich, St.Louis, MO). After staining, the sections were viewed with a fluorescentlight microscope at 20 magnification. Three images (1,349 pixels/500Am) from the distal end and three images from the proximal end werecollected and converted into JPEG format. The number of TRAP-positive cells was counted in each image. The Image J program (NIH)was used to calculate the bone area in each field.Some decalcified femurs were cryosectioned and stained foralkaline phosphatase activity (Sigma-Aldrich). The sections wereexamined with a light microscope, the images digitally collected, andanalyzed for the amount of alkaline phosphatase stain per area ofbone at the growth plate and in the trabecular region. The data werecalculated as ratio of the mm of the alkaline phosphatase stain tomm of bone.Flow cytometric and DNA analysis. Femurs were removed from five tosix mice at each time and cut into the distal and proximal metaphysesand the diaphysis. Bone marrow was flushed from these regions with a1 mL tuberculin syringe fitted with a 26-gauge needle. The marrow inthe center of the diaphysis was collected separately from the endostealmarrow close to the cortical bone as previously described (29). For flowcytometry, the RBC were lysed with ACK solution (15 mmol/L NH4Cl,1 mmol/LKHCO3, 0.1 mmol/L Na2 EDTA) and the remaining cellswere fixed with 2% paraformaldehyde. Samples were stored at 4jC untilthey were analyzed by flow cytometry (Coulter XL-MCL) usingstandardized fluorescent beads (10 Am; Sphero AccuCount RainbowFluorescent particles, Spherotech, Libertyville, IL) to estimate the totalnumber of cancer cells present. Standard curves were also generated byadding known numbers of MDA-MB-435 cells to mouse bone marrowcells. The samples of the mixtures of cells were prepared in the sameway as the experimental samples. A background value of 200 cells wasdetermined from the data obtained from the control animals in whichno GFP-positive cells were present.For DNA analysis, marrow was centrifuged and frozen inCaandMg-free Dulbecco’s PBS. At a later time, DNA was prepared from thesamples with a DNeasy kit (Qiagen, Valencia, CA). The DNA wassubjected to quantitative real-time PCR (Nucleic Acid Facility, PennState, University Park, PA) using primers to detect the HERVK gene(human endogenous retrovirus, group K), a gene found in the humanbut not in the mouse genome (30). To establish a standard curve,MDA-435 cells were counted, diluted, and added to preparations ofmouse bone marrow cells. DNA was isolated from these samples andtreated as the experimental samples for PCR. Although one cell couldbe detected in the standard curve samples, a more conservative cutoff of150 cells was used due to practical considerations, i.e., cell extractvolumes and variations in the amount of mouse DNA present in eachsample.Statistics. Each series of injections involved between 5 and 15 miceper experimental group or time. Femurs were apportioned for varioussubsequent analyses. Comparisons between groups were done by one-way ANOVA with Student-Neumann-Kuels or Tukey’s post-tests.Statistical significance was defined as a probability P V 0.05.