research article reduced latency in the metastatic niche...

Hindawi Publishing CorporationSarcomaVolume 2013 Article ID 404962 13 pageshttpdxdoiorg1011552013404962

Research ArticleReduced Latency in the Metastatic NicheContributes to the More Aggressive Phenotype ofLM8 Compared to Dunn Osteosarcoma Cells

Matthias J E Arlt Ingo J Banke Josefine Bertz Ram Mohan Ram KumarRoman Muff Walter Born and Bruno Fuchs

Laboratory for Orthopedic Research Department of Orthopedics Balgrist University Hospital University of ZurichForchstrasse 340 8008 Zurich Switzerland

Correspondence should be addressed to Matthias J E Arlt marltresearchbalgristch

Received 24 July 2013 Revised 13 October 2013 Accepted 13 October 2013

Academic Editor Akira Kawai

Copyright copy 2013 Matthias J E Arlt et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Metastasis is the major cause of death of osteosarcoma patients and its diagnosis remains difficult In preclinical studies howeverforced expression of reporter genes in osteosarcoma cells has remarkably improved the detection of micrometastases andconsequently the quality of the studies We recently showed that Dunn cells equipped with a lacZ reporter gene disseminated fromsubcutaneous primary tumors as frequently as their highly metastatic subline LM8 but only LM8 cells grew to macrometastasesIn the present time-course study tail-vein-injected Dunn and LM8 cells settled within 24 h at the same frequency in the lung liverand kidney of mice Furthermore Dunn cells also grew to macrometastases but compared to LM8 with a delay of two weeks inlung and one week in liver and kidney tissue consistent with prolonged survival of the mice Dunn- and LM8-cell-derived ovaryand spine metastases occurred less frequently In vitro Dunn cells showed less invasiveness and stronger contact inhibition andintercellular adhesion than LM8 cells and several cancer- and dormancy-related genes were differentially expressed In conclusionDunn cells compared to LM8 have a similar capability but a longer latency to form macrometastases and provide an interestingnew experimental system to study tumor cell dormancy

1 Introduction

Tumormodels inmice that make use of engrafted tumor cellsare valuable tools for preclinical research in many types ofcancer Changes in tumor phenotypes in response to geneticmanipulations in the engrafted tumor cells provide insightinto the complex mechanisms of tumor pathogenesis andmetastasis Mouse tumor models also allow the preclinicaltesting of new drugs and treatment strategies designed toimprove cancer therapy

Mouse strain-specific syngeneic or xenotransplantationmodels in immunodeficient mice have also been establishedfor osteosarcoma (OS) the most frequent primary bonetumor in children and young adults [1] A frequently usedsyngeneic OS mouse model makes use of the murine DunnOS cell line that has been isolated almost 50 years ago byDunn and Andervont from a spontaneously developing OS

primary tumor in a C3Hmouse [2] Subcutaneous (sc) rein-jection of these cells into syngeneic C3H mice revealedfast growing primary tumors but subsequent spontaneousmetastasis to the lung the predominant metastatic site inthe human disease was not observed In the late 90s AsaiandUeda succeeded in establishing a highlymetastatic Dunnsubline named LM8 [3] It was selected in vivo according tothe procedure of Poste and Fidler by serial reinjection of cellsisolated from initially rare lung metastases [4] LM8 cells incontrast to the original Dunn cells reproducibly disseminatefrom subcutaneous primary tumors to the lung and formmultiple lungmetastases with an incidence of 100 [3]Thusthe LM8 model evolved to one of the most commonly usedsyngeneic OS mouse models for preclinical drug testing [1]

We recently reevaluated the in vivometastatic propertiesof the original Dunn cells and compared it with those of thehighly metastatic LM8 subline by making use of genetically

2 Sarcoma

manipulated cells that constitutively express the bacteriallacZ gene [5] Tracking the metastasizing tumor cells indistant tissues and organs by X-gal staining down to thesingle cell level demonstrated for the first time that Dunncells spontaneously metastasize from sc primary tumorsto lungs and livers with the same incidence as LM8 cellsHowever Dunn cells different from LM8 cells did not growto macrometastatic foci and remained in the lung and theliver as micrometastases consisting of small cell clusters orsingle cells until the end of the study on day 25 Thesemicrometastases remained undetectable with standard tissuestaining techniques such as hematoxylin amp eosin stainingThese findings explain why the Dunn cells were so farconsidered as nonmetastatic

Metastasis is a complex multistep process and multiplegenes and factors regulate individual steps [6ndash8] The dif-ferent cellular processes along the metastatic cascade arealso subject to variable regulation in time including periodsof metastatic latency [9] The results of the recent studythat compared the metastatic potencies and properties ofthe LM8 and parental Dunn cells in C3H mice during anexperimental period of 25 days raised the question whethertheDunn cells lacked cellularmechanisms needed for growthand development to macrometastases in the metastatic nicheor whether they go through a longer period of metastaticlatency than the LM8 cells upon arrival in the target organs

Here metastatic properties of Dunn and LM8 cells werefurther analyzed in vitro and two follow-up studies werecarried out in vivo to answer the question raised by therecent report [5] In a first in vivo time-course study LacZ-transduced Dunn and LM8 cells were intravenously (iv)injected into the tail vein of C3Hmice and individual animalsin both groups of mice were randomly selected and sacrificedat different time points up to 25 days after tumor cellinjection In a second survival study C3H mice iv injectedwith lacZ-transduced Dunn and LM8 cells were examineduntil they became moribund and had to be sacrificed Inthe time-course study the lung liver and kidney and inthe survival study additionally the urogenital tract and insome animals the spinal cords were analyzed for micro-and macrometastases by X-gal staining of respective organsand tissues upon sacrifice Interestingly Dunn cells formedmacrometastases in the lung and in the liver and kidneywith a delay of one and two weeks respectively compared toLM8 cells The delay in the development of macrometastaseswas also reflected by significantly longer median survival ofDunn- compared to LM8-cell-injected mice In conclusionDunn cells much like LM8 cells are equipped with all thecellular mechanisms needed for dissemination to differentorgans of C3H mice but different from LM8 cells Dunncells appear to go through a period ofmetastatic latency uponarrival in these organs

2 Materials and Methods

21 Cell Lines Themouse OS cell lines Dunn and LM8 wereretrovirally transduced with a lacZ gene as described [5] andcultured at 37∘C in DMEMHam F12 (1 1) medium (Invitro-gen Carlsbad CA) supplemented with 10 heat-inactivated

FCS in an incubator with a humidified atmosphere of 5CO2

22 RNAExtraction andMicroarray Analysis RNA isolationpreparation and array hybridization were performed asdescribed [10] Briefly total RNA isolated from Dunn andLM8 cells was quantified by measuring the absorption at260 and 280 nm and RNA integrity was evaluated by agarosegel-electrophoresis Complementary RNA preparation andarray hybridization were performed by the FunctionalGenomics Center (Zurich Switzerland) using AffymetrixMouse Genome 430 20 (45101 probe sets) arrays Raw datanormalization and statistical analysis were performed usingRACE (httpraceunilch)The obtained data sets were thenfiltered by setting a fold-change cutoff gt2 (both directions)and 119875 lt 005 Resulting data were then grouped into twosets one containing the upregulated genes in LM8 comparedto Dunn and the other containing the downregulated genesBoth sets of data were then analyzed for significant enrich-ment in certain Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways via the Database for Annotation Visu-alization and Integrated Discovery (DAVID) bioinformaticsresources [11]

23 cDNA Synthesis and Real-Time PCRAnalysis 1 120583g of totalRNA was reverse-transcribed to cDNA using high capacityRNA-to-cDNA kit (Applied Biosystems Foster City CAUSA) according to the protocol provided by the manu-facturer Three independent RNA extracts from the indi-vidual cell lines were reverse-transcribed in a final vol-ume of 10 120583L Real-time PCR was carried out in StepOne-Plus Real-Time PCR System (Applied Biosystems USA)in 96-well plates Primers for Mmp2 (FP CGCTCA-GATCCGTGGTGA RP CGCCAAATAAACCGGTCC-TT) Bhlhb9 (FP CCAGCCAGAGGGAAGAATAGC RPAAAGGCAGCAGAACACAAAGC) Fn1 (FP CGAAGC-CGGGAAGAGCAAG RP CGTTCCCACTGCTGATTT-ATCTG) Src (FP TACCACTCCTCAGCCTGGAT RPACACGAGGAAGGTGGATGTC) and Gapdh (FP TGC-AGTGGCAAAGTGGAGAT RP TTTGCCGTGAGTGGA-GTCATA) were designed with the NCBI Primer BLASTsoftware (httpwwwncbinlmnihgovtoolsprimer-blast)PCRs from individual RT reactions were carried out intriplicate cDNA equivalent to 50 ng of RNA and appropriateprimers were added to Power SYBR Green PCR MasterMix (Applied Biosystems USA) and the samples were pre-incubated at 50∘C for 2min and at 95∘C for 10min and thensubjected to 40 cycles of incubation at 95∘C for 15 s and at60∘C for 1min The threshold for Ct values was set to 0325To verify the amplification of a single product in any of thePCR reactions a melting curve was generated and analyzedafter every run Relative expression levels were calculated bythe comparative cycle threshold (ΔΔCT) method and werenormalized by GAPDH expression

24 Western Blotting Cells were grown to 80 confluence innormalmedium For supernatant (SN) preparation cells werestarved over night with 2mL of serum-freemediumThenext

Sarcoma 3

day the SN was collected floating cells were removed by cen-trifugation and the volume of the SN as well as the numberof cells was determined For western blotting the volume ofthe SN was adjusted to the total number of cells SN sampleswere incubatedwith Laemmli sample buffer for 5min at 95∘Cthen separated by SDS-PAGE and subsequently transferredto nitrocellulosemembranesMMP2was detected by incuba-tion at 4∘Covernight with a rabbit polyclonal anti-MMP2pri-mary antibody (Abcam) followed by incubation at RT for 1 hwith secondary horseradish peroxidase conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology Inc) The proteinswere then visualized with Immobilon chemiluminescencesubstrate (Millipore) and quantifiedwith aVersaDoc ImagingSystem (Bio-Rad Laboratories)

25 Assessment of Intercellular Adhesion and Contact Inhibi-tion For assessing intercellular adhesion the grade of cellclustering was determined in a first assay SubconfluentDunn and LM8 cells were detached with 1mL Accutase(Sigma) counted in a hemocytometer and adjusted to250000 particles (gt90 single cells) permL Two mL werethen seeded into 6-well ultralow attachment plates (10 cm2Costar) and four pictures per well of random fields weretaken with a 4x objective (36mm2) and a Zeiss ObserverZ1microscope immediately after seeding and at different timepoints thereafter Particle number analysis of pictures wasperformed using ImageJ Clustering results in a decreaseof particle number After 25 h clusters were dissociated bypipetting 20 times up and downwith a 1mLGilson pipetThepercent of clusters of gt1000 pixels over total particles (gt20pixels) was then calculated In a second assay intercellularadhesion was estimated by counting the number of singlecells and of clusters of two or three cells in suspensions oftrypsinized subconfluent and confluent cells The percentageof single cells was calculated from the number of single cellsand of cell clusters observed in three randomly selected fieldsof the hemocytometerThe total number of single cells or cellclusters counted in individual experiments varied between100 and 150 To estimate the strength of intercellular adhesioncells were seeded in 24-well plates at between 30 and 40confluence and allowed to form a confluent monolayer Sixwounds per cell line and experiment were then generatedwith a sterile pen with a tip diameter below 1mm Thewound widths were then measured using a Nikon DiaphotTMD microscope with a 10x objective and a 10x ocular witha graded 1mm scale Contact inhibition was assessed bycounting the total number of cells grown to subconfluenceor visual confluence in 25 cm2 tissue culture flasks Thecells were trypsinized and aliquots of cell suspensions werecounted in a hemocytometer

26 Three-Dimensional Matrigel Degradation and MigrationAssay Subconfluent cells were trypsinized and resuspendedin ice-cold cell culture medium without FCSThe cell densitywas estimated with a hamocytometer and adjusted to 500cells per120583L Equal volumes of ice-cold cell suspension andMatrigel (10mgmL) were mixed and 25 120583L were appliedto the center of a 6-well plate in triplicate After gelling at

37∘C for 30min 2mL of cell culture medium supplementedwith 10 FCS was added Evasion foci outside the theMatrigel boarders which resulted from combined migrationand degradation of Matrigel matrix were counted under amicroscope Evasion events per drop were proportional tothe number of cells per drop and evasion occurred earlier athigher cell densities of seeded cells (not shown)

27 Time Course of Dunn and LM8 Experimental Metasta-sis Eight-week-old female C3HHeNCrl (C3H) mice wereobtained from Charles River Laboratories (Sulzfeld Ger-many) at least 10 days before the beginning of the experimentHousing conditions and experimental protocols were inaccordance with the guidelines of the ldquoSchweizer Bundesamtfur Veterinarwesenrdquo and approved by the authorities of theCanton of Zurich On day 0 of the experiment 106 lacZ-transduced Dunn or LM8 cells in 200 120583L PBS were injectedinto the tail vein of the mice The health of the mice wasmonitored daily On days 1 4 7 14 21 and 25 after tumorcell injection 4 randomly selected mice per group weresacrificed and selected tissues and organs were analyzed forthe presence of metastases as recently described [5] Brieflyblood was removed from the lungs by perfusion with PBSunder anesthesia with ketamine xylazine and acepromazineThe lungs were then fixed in situ for 10min under inflationwith 3 paraformaldehyde (PFA) Subsequently the lungslivers and kidneys were removed fixed with 2 PFA and02 glutaraldehyde in PBS at room temperature for 30minand then washed with PBS and stained with X-gal solution(Enzo Life Sciences AG Lausen Switzerland) at 37∘C forat least 3 hours Photographs of whole lungs and liverswere taken with an E620 DSLR camera under an SZX 10binocular microscope (Olympus Corporation Tokyo Japan)and imported as JPEG files into PowerPoint software Close-ups of organ surfaces were taken with a Kappa PS 20 C digitalcamera (Kappa opto-electronics GmbH Gleichen Germany)attached to an Eclipse E600microscope (Nikon CorporationTokyo Japan) and imported as TIFF files into PowerPointsoftware Due to the great convexity and intense colouring ofthe kidney close-ups of micrometastases on its surface couldnot be taken

28 Survival Experiment with the Dunn and LM8 Experi-mental Metastasis Models The mouse strain and source thehousing conditions the number of tumor cells and the routeof tumor cell injection were those described above In thisexperiment the metastases-bearing mice (8 per group) werekept alive as long as possible and were sacrificed individuallywhen they became moribund The lung liver and kidneys aswell as other selected organs (ovaries spine) were then alsoprepared and analyzed as described

29 Statistical Analysis The in vitro data were statisticallyanalyzed with the two-tailed paired studentrsquos 119905-test and theKaplan-Meier survival curves were statistically analyzed withthe log rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test Data were considered significantly differentwhen 119875 lt 005

4 Sarcoma

3 Results

31 Differences between Dunn and LM8 Cells in IntercellularAdhesion Contact Inhibition and Invasiveness In Vitro Lossof intercellular adhesion and contact inhibition as well as theability to degrade and migrate through extracellular matrixare prerequisites for metastasizing tumor cells to colonizedistant organs In the present study we found that afterseeding the same number of gt90 solitary cells in ultralowattachment plates the Dunn cells (upper panel in Figure 1(a))are aggregated much faster than the LM8 cells (lower panelin Figure 1(a)) to clusters resulting in a significantly higher(119875 lt 005) aggregation rate within the first two hours(Figure 1(b)) After 1 day both cell lines had formed roundedclusters with mean areas of 3509 plusmn 547 120583m2 for Dunn and3188 plusmn 373 120583m2 for LM8 which further increased signifi-cantly until day 3 to 6065 plusmn 800 120583m2 (119875 lt 0002) and 5241 plusmn576 120583m2 (119875 lt 004) Interestingly after 1 and 3 days the clus-ters formed by Dunn cells were more regular (round spheres)than those formed by the LM8 cells (Figure 1(a)) Dissocia-tion of clusters after one day (last picture in both panels ofFigure 1(a)) revealed a lower amount (119875 lt 0004) of bigclusters in LM8 compared to Dunn (Figure 1(c)) Further-more when the percentage of single cells was assessed intrypsinized populations derived from confluent Dunn andLM8 cells it was found to be 17 higher (119899 = 5 119875 lt0003) in LM8 than in Dunn cultures (data not shown)These observations suggested that in confluent culturesintercellular adhesion between Dunn cells was stronger thanthat between LM8 cells These observations were also inagreement with those made in a wounding assay There thewidths of wounds applied to LM8 cell monolayers were 32(119899 = 6 119875 lt 00002) smaller than those found in woundsof Dunn cell monolayers (Figure 1(d)) indicating again asignificantly decreased strength in intercellular adhesionbetween LM8 compared to Dunn cells In addition the celldensity of LM8 cells at visual confluence was 70 higher thanthat of Dunn cells (119899 = 5 119875 lt 0005) implying a significantloss of contact inhibition of LM8 compared to Dunn cells(Figure 1(e))

The ability of Dunn and LM8 cells to degrade and tomigrate in a three-dimensional matrix was evaluated witha Matrigel drop assay Immediately and up to 24 h aftercell seeding cells that had migrated out of the Matrigeldrops were not observed (Figure 1(f)) At later time pointsevasion foci were visible on the surface of the Matrigel dropsindicating that the OS cells had locally degraded Matrigeland had migrated to and slightly beyond the surface of thedrop (Figure 1(g)) The number of evasion foci continuouslyincreased up to 96 h (Figure 1(h)) After longer incubationperiods adjacent foci began to fuse and were no longerquantifiable Two days after cell seeding the number of LM8evasion foci increasedmuch faster than that ofDunn cells andthe largest differencewas observed at 72 hwhenLM8cells hadformed a 6 times higher number of evasion foci than Dunncells (119875 lt 0003 Figure 1(i)) The results indicate a highermobility and matrix degrading capacity of LM8 compared toDunn cells

Table 1 Number of regulated (gt2-fold P lt 005) DunnLM8 genesets enriched in KEGG pathways (DAVID)

Pathways Gene count 1

Pathways in cancer 31 378Focal adhesion 22 268Cytokine-cytokine receptor interaction 19 231Regulation of actin cytoskeleton 16 195Axon guidance 14 171Cell adhesion molecules (CAMs) 14 171Vascular smooth muscle contraction 11 134ECM-receptor interaction 10 122Apoptosis 9 110Basal cell carcinoma 8 097Complement and coagulation cascades 8 097Adherens junction 8 097Hematopoietic cell lineage 8 097Hedgehog signaling pathway 7 085Glutathione metabolism 6 073Prion diseases 5 061Glycosphingolipid biosynthesis 4 0491Proportion of all regulated gene sets

32 Gene Expression Analysis of Dunn and LM8 Cell LinesRevealsDifferentially RegulatedGenes in ldquoPathways inCancerrdquo(KEGGPathway) and amongDormancyGenes There is goodevidence that disseminated tumor cells which have acquiredall necessary properties to mature to macrometastatic foci inthe metastatic niche of distant organs may preserve thoseproperties when they are isolated and cultured in vitro [9 12]Based on this assumption we performedmicroarray analysisof the Dunn and LM8 cell lines and subsequently analyzedthe differences in their gene expression profiles In total 1257gene sets were differentially regulated (gt2-fold change 119875 lt005) in Dunn and LM8 cells The DAVID program detectedenrichment of these regulated gene sets in 17 KEGG path-ways (Table 1) The highest gene enrichment was found forldquoPathways in Cancerrdquo (378) followed by ldquoFocal Adhesionrdquo(268) ldquoCytokine-Cytokine Receptor Interactionrdquo (231)and ldquoRegulation of Actin Cytoskeletonrdquo (195) From the 31gene sets that were associated with cancer pathways 19 weresignificantly down- and 12 significantly upregulated in thehighly metastatic LM8 cells compared to the low-metastaticDunn cells (Table 2) Interestingly the downregulated geneswere not only predominant in number but also with regard tofold change While the upregulation in LM8 cells was highestfor matrix metallopeptidase 2 (Mmp2) gene transcripts (sim7-fold) the most pronounced downregulation (sim24-fold) wasfound for kit ligand (Kitl) gene transcripts The fold changein the top 5 of the downregulated genes (Kitl Pparg VegfcPtgs2 and Pdgfb) ranged from 2457 to 595 and in the top5 of the upregulated genes (Mmp2 Dapk2 Tcf7 Wnt1 andBirc3) from 691 to 318

In a second analysis we checked our microarray data fordifferential regulation of genes that are likely involved in

Sarcoma 5

0h 2h 25h 67h 25h + dissD

unn

LM8

(a)

0 60 1200

50

100

DunnLM8

Time (min)

Part

icle

s (

tim

e zer

o) lowast lowast

lowast

(b)

Dunn LM800

05

10

15

20

25

Part

icle

s (gt1000

pixe

ls

)

lowast

(c)Dunn LM8

0

200

400

600

800

Wou

nd w

idth

(120583m

)

lowast

(d)Dunn LM8

00

02

04

06

Cell

scm

2(times10

6)

lowast

(e) (f) (g)

0 20 40 60 80 1000

10

20

30

LM8Dunn

Time (h)

Evas

ion

even

ts

lowast

lowast

(h)

Dunn LM80

5

10

15

20

Evas

ion

even

ts

lowast

(i)

Figure 1 Intercellular adhesion contact inhibition and invasiveness of Dunn and LM8 cells in vitro (a) Representative time course ofintercellular adhesion and dissociation of Dunn (upper panels) and LM8 (lower panels) Scale bar 400120583m (b) Quantification of particlenumber in percent of the 0min value Particle numbers at time zero were 1036plusmn105 and 1313plusmn93 (119875 gt 005) in Dunn and LM8 respectivelyand were set to 100 (c) Dissociation by pipetting after 25 h association Results are the means plusmn SEM of 3 independent experiments (d)Intercellular adhesion was further estimated by wounding of confluent cells grown in 24-well plates with a pen and subsequent measurementof the width of the fresh wound as described in Section 2 (e) The cells were grown in 25 cm2 flasks to visual confluence and then trypsinizedand counted to estimate differences in contact inhibitionThe invasive activity of cells in vitrowas determined in a three-dimensionalMatrigeldegradation and migration assay Representative microscopic images of LM8 cells seeded in Matrigel drops (500 cells120583L) immediately aftergelling (f) and after incubation for 72 h in cell culture medium (g) are shown (scale bars in (f) and (g) 500 120583m) Bold arrows in (f) and (g)point to the border of the Matrigel drop and normal arrows in (g) to foci evading from the Matrigel drop from cell clusters growing insidetheMatrigel drop (h)The number of foci evading from theMatrigel drops (119899 = 3) was counted daily (i)The difference between the numbersof Matrigel-evading Dunn and LM8 cell foci per drop (119899 = 4) peaked at 72 h after cell seeding All results are shown as the means plusmn SEM of3ndash6 independent experiments Asterisks (lowast) indicate statistically significant difference (119875 lt 005)

tumor cell dormancy [13ndash15] For 7 out of 44 investigatedgenes (described in [13ndash15]) the expression was significantlydifferent between Dunn and LM8 cells (gt2-fold change 119875 lt005 Table 3) Among those genes the connective tissue

growth factor (Ctgf) genewas the only one found upregulated(272-fold) in LM8 compared to Dunn cells Basic helix-loop-helix domain containing class B9 (Bhlhb9) with a 61-folddecrease was one of the most heavily downregulated genes in

6 Sarcoma

0

1

2

3

420

40

60

80

DunnLM8

Mmp2 Bhlhb9 Fn1 Src

2(minusΔΔ

Ct)

Microarray 69 minus61 minus29 minus37 (LM8Dunn)

(a)

LM8

Dun

n

LM8

Dun

n

SN 2SN 1

MMP2

(b)

Dunn LM80

5

10

Fold

chan

ge

(c)

Figure 2 (a) qRT-PCR analysis of Mmp2 Bhlhb9 Fn1 and Src mRNA expression normalized to Gapdh in Dunn and LM8 cells Forcomparison the fold-change values calculated from the microarray data are indicated above the graph (b) Representative western blotsof MMP2 protein levels in FCS-free supernatant of Dunn and LM8 cells and (c) quantitative analysis of three independent western blotsexperiments Results are shown as the means plusmn SEM

LM8 compared to Dunn cells The fibronectin 1 (Fn1) Roussarcoma oncogene (Src) transforming growth factor beta 2(Tgfb2) transforming growth factor beta receptor 1 (Tgfbr1)and tropomyosin 1 alpha (Tpm1) genes on the other handwere downregulated only 2-3-fold in LM8 compared toDunncells

For selected genes (Mmp2 Bhlhb9 Fn1 and Src) the dataof the microarray were confirmed by qRT-PCR (Figure 2(a))ForMmp2 Bhlhb9 andFn1 the difference in expression of theindividual genes betweenDunn and LM8 cells was evenmorepronounced than in themicroarray analysis Furthermore theprotein expression of MMP2 was analyzed in conditionedmedium The western blot analysis revealed 8-fold higherMMP2 levels in FCS-free supernatant of LM8 cells than insupernatant of Dunn cells (Figures 2(b) and 2(c))

33 Delayed Formation of Macrometastases by Dunn Com-pared to LM8 Cells in C3H Mice Is Associated with ProlongedSurvival Potential differences in the outgrowth of Dunn-

and LM8-cell-derived lung metastases were investigated ina time-course study that monitored the formation of micro-and macrometastases over time in selected organs of C3Hmice upon iv injection of 1 times 106 LacZ-transduced Dunn orLM8 cells Micrometastases of both cell lines consisting ofsingle cells or cell clusters smaller than 01mm in diameterwere already observed one day after iv tumor cell injection(TCI) in the lungs (Figure 3(a) first set of close-up imagesin the upper and lower panels) and on the liver (Figure 3(b))and kidney (Figure 3(c)) surfaces On day 4 after tumor cellinjection the metastatic pattern in the organs of both Dunnand LM8-cell-injected mice appeared unchanged Howeveroneweek after TCI the firstmetastatic foci larger than 01mmconsidered as macrometastases became visible in the lungsof mice injected with LM8 cells (Figure 3(a) lower panel)Macrometastases in the livers and kidneys of LM8-injectedmice were first detected in animals that were sacrificed twoweeks after TCI (lower panels in Figures 3(b) and 3(c)) Inmice injected with Dunn cells on the other hand analyzed

Sarcoma 7

Day 1 Day 4 Day 7 Day 14 Day 21 Day 25

Dunn

LM8

(a)

Dunn

LM8


(b)

Dunn

LM8


(c)

Figure 3 Continued

8 Sarcoma

First micromets in

lung liver and kidney

First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)

Figure 3 Appearance over time of experimental metastases in indicated organs after intravenous injection of lacZ-transduced Dunn andLM8 cells in C3Hmice ((a)ndash(c)) Images show X-gal-stained metastases in blue in representative whole mounts and ((a) (b)) correspondingclose-ups of lungs (a) livers (b) and kidneys (c) collected from mice that were sacrificed at indicated time points after tumor cell injection(d) Schematic summary of the first appearance of micro- and macrometastases (gt01mm) over time in indicated organs

organs still contained only micrometastases at the end ofweeks one and two of the experiment (upper panels inFigures 3(a)ndash3(c)) But interestingly three weeks after Dunncell injection the lungs (Figure 3(a) upper panel) liversand kidneys (upper panels in Figures 3(b) and 3(c)) of themice showed for the first time outgrowing macrometastasesHowever these Dunn-cell-derived macrometastases wereconsiderably smaller than those observed at the same timein the lungs livers and kidneys of LM8-injected miceApparently comparable continuous growth of respectivemacrometastases maintained the observed differences in sizeuntil the end of the study on day 25 Interestingly in both theDunn and the LM8-cell-injected C3H mice the final size ofrespective liver and kidney macrometastases was larger thanthat of the respective lung macrometastases In LM8-injectedmice this was an unexpected observation because as notedbefore macrometastases in the livers and kidneys devel-oped with one week delay compared to those in the lungsFigure 3(d) schematically summarizes the results obtained inthis time-course study

The additional survival study was performed to investi-gate the effect of the delayed outgrowth of Dunn-comparedto LM8-cell-derived macrometastases on the survival of therespective mice C3H mice were iv injected with the samenumber of tumor cells as in the time-course study andsacrificed when they became moribund Figure 4(a) showsthe corresponding Kaplan-Meier curves In the group ofmiceinjectedwith LM8 cells the first 3 animals had to be sacrificedon day 25 after TCI and the median survival time in thisgroup was 26 days The last two mice of this group becamemoribund on day 33 The first mice in the group of animalsinjected with Dunn cells on the other hand had to be killedon day 32 and the median survival time was 40 days Thelast mouse in this group was sacrificed on day 85 Overallthese results revealed a statistically significant difference (119875 lt0005) in the survival of the two groups of mice Analysisof the inner organs of moribund Dunn- and LM8-injected

mice confirmed the faster growth to macrometastases of thetwo cell lines in the liver (Figure 4(b) (iii-iv)) and kidney(Figure 4(b) (v-vi)) than in the lung (Figure 4(b) (i-ii)) Inaddition massive metastases were also found in severalovaries of Dunn-LacZ- (Figure 4(b) (v)) and LM8-LacZ-injected mice (Figure 4(b) (vi)) Furthermore a few mice ofboth groups showed paralysis of the hindlimbs Postmortemanalysis of their spinal columns revealed metastases in thevertebral bodies (Figure 4(b) (vii-viii)) Some of these metas-tases were so large that they had already infiltrated the lumenof the spine leading to compression of the spinal cord whichwas most likely the cause of the paralysis

4 Discussion

Metastases are the major cause of death in OS A bet-ter understanding of the cellular mechanisms involved inthe multistep metastatic processes and early diagnosis ofmetastatic disease are therefore of importance for an adequatetherapy on time and might result in improved survival ofthe patients Unfortunately although all disseminating tumortypes employ largely similar mechanisms in the multistageprocess of local tissue invasion intravasation survival inthe circulation extravasation and colonization of distantorgans their disseminating activities differ considerably infrequency direction and temporal course Even within thesame type of cancer these characteristics can differ dependingon the genetic changes acquired by the tumor cells during themetastatic process [9 12]

Recently we have been able to demonstrate that differentfrom what was initially reported sc injected mouse DunnOS cells disseminate to the lung and liver with the samefrequency as their highly metastatic derivatives LM8 butdifferent from LM8 cells Dunn cells did not further developto macrometastases during the experimental period of 25days [5] Based on this novel so far not reported disseminatingphenotype of the DunnOS cells we performed in the present

Sarcoma 9

Table 2 Genes involved in cancer pathways (KEGG pathway DAVID) and whose expression is significantly down- or upregulated in LM8compared to Dunn cells

Symbol Fold change P valueGenes downregulated in LM8 cells

Kit ligand Kitl minus2457 426119864 minus 12

Peroxisome proliferator activated receptor gamma Pparg minus1570 202119864 minus 10

Vascular endothelial growth factor C Vegfc minus820 350119864 minus 09

Prostaglandin-endoperoxide synthase 2 Ptgs2 minus746 434119864 minus 08

Similar to platelet-derived growth factor B chain platelet-derived growth factor B polypeptide Pdgfb minus595 136119864 minus 09

Insulin-like growth factor 1 Igf1 minus470 388119864 minus 08

Aryl hydrocarbon receptor nuclear translocator 2 Arnt2 minus380 116119864 minus 05

Endothelial PAS domain protein 1 similar to endothelial PAS domain protein 1 Epas1 minus362 113119864 minus 05

Fibronectin 1 Fn1 minus295 569119864 minus 08

Integrin alpha 3 Itga3 minus279 102119864 minus 07


Laminin B1 subunit 1 Lamb1-1 minus264 826119864 minus 05

Transforming growth factor beta 2 Tgfb2 minus243 609119864 minus 06

RAS-related C3 botulinum substrate 3 Rac3 minus237 165119864 minus 04

Bone morphogenetic protein 4 Bmp4 minus226 864119864 minus 07

Signal transducer and activator of transcription 3 similar to Stat3B Stat3 minus223 564119864 minus 06

Transforming growth factor beta receptor I Tgfbr1 minus215 506119864 minus 06

Fibroblast growth factor receptor 2 Fgfr2 minus214 969119864 minus 07

Interleukin 6 Il6 minus201 578119864 minus 05

Genes upregulated in LM8 cellsMatrix metallopeptidase 2 Mmp2 691 424119864 minus 09

Death-associated protein kinase 2 Dapk2 470 779119864 minus 10

Transcription factor 7 T-cell specific Tcf7 382 121119864 minus 06

Wingless-related MMTV integration site 1 Wnt1 324 255119864 minus 07

Baculoviral IAP repeat-containing 3 Birc3 318 257119864 minus 08

Patched homolog 2 Ptch2 302 166119864 minus 05

Frizzled homolog 7 (Drosophila) Fzd7 294 775119864 minus 07

Wingless-related MMTV integration site 10a Wnt10a 264 193119864 minus 05

Fibroblast growth factor 15 Fgf15 263 279119864 minus 05

GLI-Kruppel family member GLI2 Gli2 259 162119864 minus 05

Similar to wingless-related MMTV integration site 8b wingless-related MMTV integration site 8b Wnt8b 239 389119864 minus 04

Predicted gene 10124 predicted gene 6340 CDC28 protein kinase 1b Cks1b 229 124119864 minus 06

Genes with gt2 times decrease or increase in expression and a P value lt 005 were selected and applied to the DAVID program

study experiments in vitro and in vivo to further characterizethe metastatic capabilities of Dunn compared to LM8 cellsMetastatic properties such as contact inhibition intercellularadhesion and invasiveness were examined in vitro In vivotime-course and survival studies were designed to answer thequestion whether Dunn cells that infiltrate lung or liver tissuewill never grow tomacrometastases or even disappear or onlyremain latent for a certain time as single cells or small cellclusters before they restart excessive proliferation and formovert metastases as it has been described for for examplebreast cancer cells [16 17]

The results of the in vitro studies revealed indeed aputative milder metastatic phenotype for the Dunn cellsfor example less invasiveness and stronger contact inhibi-tion and intercellular adhesion than observed for the LM8cells Interestingly the here observed significantly higher

aggregation rate and faster formation of spheres of Dunncompared to LM8 cells in ultralow attachment plates arein good accordance with experiments in soft agar assaysin the previous study which indicated a faster anchorageindependent growth of Dunn compared to LM8 cells [5]These findings taken together suggest that factors other thanproliferation rates might determine the metastatic growthof the two cell lines This is in agreement with anotherstudy where LM8 cells overexpressing different cadherinsshowed in comparison to the wild type LM8 massivelyreduced formation of lung metastases in vivo but similarproliferation rates in vitro [18]The differences in invasivenessbetween the Dunn and LM8 cells observed in the presentstudy in the Matrigel drop assay are in agreement withthe findings of Asai et al who demonstrated higher MMP-2 expression and activity in LM8 than in Dunn cells [3]

10 Sarcoma

Table 3 Regulation of potential dormancy genes in LM8 compared to Dunn cells

Symbol Fold change P valueGenes downregulated in LM8

Basic helix-loop-helix domain containing class B9 Bhlhb9 minus6098 846119864 minus 13Fibronectin 1 Fn1 minus295 569119864 minus 08


Rous sarcoma oncogene Src minus236 224119864 minus 06

Tropomyosin 1 alpha Tpm1 minus228 253119864 minus 07


Genes upregulated in LM8Connective tissue growth factor Ctgf 272 218119864 minus 04

Genes not differentially regulated in LM8Secreted phosphoprotein 1 Spp1 minus178 208119864 minus 05

A kinase (PRKA) anchor protein (gravin) 12 SSeCKS Akap12 minus167 518119864 minus 05

plasminogen activator urokinase receptor uPAR Plaur minus159 380119864 minus 04

Cyclin-dependent kinase inhibitor 2B (p15 inhibits CDK4) INK4b p15 Cdkn2b minus147 466119864 minus 03

Discoidin domain receptor family member 2 Ddr2 minus146 214119864 minus 03

Angiomotin Amot minus116 807119864 minus 02

Thrombospondin 1 Thbs1 minus113 148119864 minus 01

Nuclear receptor subfamily 2 group F member 2 Nr2f2 minus110 262119864 minus 01

Basic helix-loop-helix family member e41 Sharp1 Bhlhb3 Bhlhb2l Bhlhe41 minus109 222119864 minus 01

Smad family member 7 Smad7 minus107 507119864 minus 01

NMENM23 nucleoside diphosphate kinase 1 Nme1 minus103 695119864 minus 01

Insulin-like growth factor binding protein 5 IGFBP5 minus103 756119864 minus 01

Integrin alpha 5 (fibronectin receptor alpha) Itga5 minus102 871119864 minus 01

Mitogen-activated protein kinase kinase 7 MKK7 Map2k7 minus101 948119864 minus 01

Rho GDP dissociation inhibitor (GDI) beta RhoGD12 Arhgdib minus101 905119864 minus 01

Mitogen-activated protein kinase 14 p38 Mapk14 minus101 866119864 minus 01

Eph receptor A5 EphA5 minus101 906119864 minus 01

Integrin beta 1 (fibronectin receptor beta) Itgb1 101 952119864 minus 01

Transformation related protein 53 p53 Trp53 102 707119864 minus 01

Endothelial cell-specific molecule 1 Esm1 103 689119864 minus 01

Mitogen-activated protein kinase kinase 4 MKK4 Map2k4 104 706119864 minus 01

Tissue inhibitor of metalloproteinases 3 Timp3 104 682119864 minus 01

Eph receptor B2 Erk Ephb2 105 747119864 minus 01

CD82 antigen Kai1 Cd82 106 401119864 minus 01


51015840 Nucleotidase ecto CD73 Nt5e 108 420119864 minus 01

Epidermal growth factor receptor Egfr 111 353119864 minus 01

Phosphatidylinositol 3-kinase catalytic beta polypeptide PI3K (Pik3cb) 112 309119864 minus 01

Twist basic helix-loop-helix transcription factor 2 Twist2 113 130119864 minus 01

Mediator complex subunit 23 CRSP3 Med23 115 753119864 minus 02

Lysophosphatidic acid receptor 1 Edg2 Lpar1 119 146119864 minus 02

Breast cancer metastasis-suppressor 1-like Brms1l 127 276119864 minus 02

GATA binding protein 3 Gata3 134 145119864 minus 02

Procollagen-proline 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase) alpha 1 polypeptide P4ha1 175 428119864 minus 05

Genes with gt2 times decrease or increase in expression and a P value lt 005 were considered as significantly regulated For Igf1r Kiss1 and Hist1h2bk no datawere available on the microarray

which we were able to confirm in the present study with ourmicroarray qRT-PCR andwestern blot data It is conceivablethat the different capabilities of Dunn and LM8 cells todegrade and penetrate extracellular matrix might be more

important for infiltration and colonization of target organsthan for the escape from the primary tumor At the primarytumor site cancer cells are usually quite well supportedby recruited stromal and immune cells which substantially

Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)

(b)Figure 4 Animal survival and tissue distribution of metastases atsacrifice in mice intravenously injected with lacZ-transduced Dunnor LM8 OS cells (a) Kaplan-Meier survival curves of C3H miceinjected with LM8 (red line) or Dunn (blue line) cells (b)Metastaticpattern in lung (i-ii) liver (iii-iv) urogenital tract (v-vi) and spine(vii-viii) of moribund mice at sacrifice

contribute to for example local degradation of the ECM byexpressing several proteases [19 20] In distant organs theymight be much more dependent on their own capabilities toinvade healthy tissue However the ability of disseminatingtumor cells to invade the tissue of distant organs is onlya prerequisite but not sufficient for the formation of overtmetastases It is known that even certain premalignant celllineages have the capacity to disseminate infiltrate distantorgans and survive in tissues different from their origin [21]Tumor cells therefore have to acquire additional propertiesto achieve full metastatic competence [9] Our microarraydata indicated that under in vitro culture conditions 31 genesassociated with cancer pathways and 7 genes involved intumor cell dormancy were differentially regulated in thelow-metastatic Dunn compared to the highly metastaticLM8 cells Interestingly almost 23 of the genes involvedin cancer pathways and 6 out of 7 dormancy-related geneswere expressed at lower levels in the highly metastatic LM8than in the low-metastatic Dunn cells suggesting that thedownregulation of certain genes is of equal or even higherimportance than the upregulation of others This is furthersupported by the observation that the fold decrease of manydownregulated genes was much higher than the fold increaseof most upregulated genes Bhlhb9 was among the top 7most heavily regulated genes recognized in this microarrayanalysis Its product belongs to the basic helix-loop familyof transcription factors which is involved in tumorigenesis-associated gene regulation [22] Members of this family forexample BHLHB3 induce growth arrest of disseminatedtumor cells without suppressing primary tumor growth [23]and BHLHB9 itself might play a pivotal role in apoptotic celldeath [24] PPARG also strongly downregulated in LM8 cellsbelongs to the nuclear hormone receptor superfamily andas a tumor repressor gene it is silenced or mutated duringtumorigenesis of for example sporadic colorectal cancerand its down-regulationinactivation correlates significantlywith the aggressiveness of the disease [25] Interestinglythe expression of quite a few angiogenesis- and metastasis-related genes particularly growth factors was also founddownregulated in LM8 compared to Dunn cells It is knownthat many cancer-associated gene products have bifunctionalroles in tumor progression and metastasis for exampleTGF1205732 and TGF120573R1 TGF120573 is often prominently expressedin the tumor microenvironment and promotes beside tumorgrowth also metastasis by inducing the expression of genesimportant for the dissemination of tumor cells [26] Indistant organs on the other hand its autocrineparacrinesignaling might by considerably involved in induction andmaintenance of dormancy in disseminated tumor cells [14]

The results of the in vivo experimental metastasis studiesare in good agreement with these microarray results and thefindings obtained in vitro since they showed that the Dunncells much like the LM8 cells are capable of growing tomacrometastases in the lung the liver and the kidney and insome mice even in ovaries and vertebral bodies of the spinebut with a considerable delay when compared to LM8 cellsThus the Dunn OS cells are not only able to disseminatefrom primary tumors to distant organs as reported in theprevious study [5] but also grow to macrometastases but

12 Sarcoma

during a phase of dormancy they need to acquire some addi-tional properties in the new tissuemicroenvironments In thiscontext it is also important to note that in LM8-cell-injectedmice lung macrometastases occurred one week earlier thanthose recognized in the livers and kidneys despite the factthat all these organs contained micrometastases as early asone day after iv injection of LM8 cells This indicates thatthe LM8 cells that were initially selected for a high lungmetastatic potential apparently needed some extra time toadapt to the liver or kidney microenvironments before theystarted to grow tomacrometastases in these organsTheymayhave acquired a lung-specific tropism during their generationby several cycles of in vivo selection of Dunn-cell-derivedlung metastases Such organ-specific metastatic phenotypeshave been reported and can also be stably maintained ex vivo[12] However detailed molecular mechanisms that provide alung-specific tropism remain to be investigated Surprisinglyat the end of the study liver and kidney macrometastasesin both the Dunn and the LM8-cell-injected mice were byfar larger in size than the respective lung metastases Theseliver and kidney macrometastases besides occasional ovaryand spine metastases were likely also the life-threateningmetastases for the mice investigated in the survival study

Altogether the results of the in vivo studies unequivocallyshowed that the Dunn OS cells are also capable of growing tomacrometastases in different tissue environments but withsome latency when compared to LM8 cells which resultedin prolonged survival The results further indicate thatafter acquisition of a potentially organ-specific colonizationcompetence both the Dunn and LM8 OS cells proliferatedunrestrictedly in the different organs

In conclusion the results of the present study provideevidence for faster adaptation of the highly metastatic mouseLM8 OS cells than of the parental Dunn OS cells to therequirements for colonization of distant tissues after dissemi-nation from the primary tumors Apart from that Dunn andLM8 OS cells exhibit a comparable metastatic phenotype insyngeneic C3H mice Consequently the Dunn OS cell linecan no longer be considered as non- or low metastatic andit provides an interesting new experimental system to studytumor dormancy

Conflict of Interests

The authors state no conflict of interests

Authorsrsquo Contribution

Matthias J E Arlt and Ingo J Banke contributed equally tothis work

Acknowledgments

This work was supported by grants from the Krebsliga of theCanton of Zurich the Walter L and Johanna Wolf Foun-dation Zurich the Lydia Hochstrasser Foundation Zurichthe Swiss National Science Foundation SNF Switzerland

the Schweizerischer Verein Balgrist and the University ofZurich

References

[1] E T H Ek C R Dass and P F M Choong ldquoCommonlyused mouse models of osteosarcomardquo Critical Reviews inOncologyHematology vol 60 no 1 pp 1ndash8 2006

[2] T B Dunn andH B Andervont ldquoHistology of some neoplasmsand non-neoplastic lesions found in wild mice maintainedunder laboratory conditionsrdquo Journal of the National CancerInstitute vol 31 pp 873ndash901 1963

[3] T Asai T Ueda K Itoh et al ldquoEstablishment and char-acterization of a murine osteosarcoma cell line (LM8) withhigh metastatic potential to the lungrdquo International Journal ofCancer vol 76 no 3 pp 418ndash422 1998

[4] G Poste and I J Fidler ldquoThe pathogenesis of cancermetastasisrdquoNature vol 283 no 5743 pp 139ndash146 1980

[5] M J E Arlt I J Banke D K Walters et al ldquoLacZ trans-gene expression in the subcutaneous DunnLM8 osteosarcomamouse model allows for the identification of micrometastasisrdquoJournal ofOrthopaedic Research vol 29 no 6 pp 938ndash946 2011

[6] A C Chiang and J Massague ldquoMolecular basis of metastasisrdquoTheNew England Journal of Medicine vol 359 no 26 pp 2814ndash2823 2008

[7] G P Gupta and J Massague ldquoCancer metastasis building aframeworkrdquo Cell vol 127 no 4 pp 679ndash695 2006

[8] D X Nguyen and J Massague ldquoGenetic determinants of cancermetastasisrdquo Nature Reviews Genetics vol 8 no 5 pp 341ndash3522007

[9] D X Nguyen P D Bos and J Massague ldquoMetastasis fromdissemination to organ-specific colonizationrdquo Nature ReviewsCancer vol 9 no 4 pp 274ndash284 2009

[10] R Muff R M Ram Kumar S M Botter W Born and BFuchs ldquoGenes regulated inmetastatic osteosarcoma evaluationby microarray analysis in four human and two mouse cell linesystemsrdquo Sarcoma vol 2012 Article ID 937506 13 pages 2012

[11] D W Huang B T Sherman and R A Lempicki ldquoSystematicand integrative analysis of large gene lists using DAVID bioin-formatics resourcesrdquo Nature Protocols vol 4 no 1 pp 44ndash572009

[12] A J Minn Y Kang I Serganova et al ldquoDistinct organ-specificmetastatic potential of individual breast cancer cells andprimary tumorsrdquo Journal of Clinical Investigation vol 115 no 1pp 44ndash55 2005

[13] D Paez M J Labonte P Bohanes et al ldquoCancer dormancya model of early dissemination and late cancer recurrencerdquoClinical Cancer Research vol 18 no 3 pp 645ndash653 2012

[14] P Bragado M S Sosa P Keely J Condeelis and J A Aguirre-Ghiso ldquoMicroenvironments dictating tumor cell dormancyrdquoRecent Results in Cancer Research vol 195 pp 25ndash39 2012

[15] N Almog L Ma R Raychowdhury et al ldquoTranscriptionalswitch of dormant tumors to fast-growing angiogenic pheno-typerdquo Cancer Research vol 69 no 3 pp 836ndash844 2009

[16] Y Husemann J B Geigl F Schubert et al ldquoSystemic spreadis an early step in breast cancerrdquo Cancer Cell vol 13 no 1 pp58ndash68 2008

[17] O Schmidt-Kittler T Ragg A Daskalakis et al ldquoFrom latentdisseminated cells to overt metastasis genetic analysis ofsystemic breast cancer progressionrdquo Proceedings of the National

Sarcoma 13

Academy of Sciences of the United States of America vol 100 no13 pp 7737ndash7742 2003

[18] T Kashima K Nakamura J Kawaguchi et al ldquoOverexpressionof cadherins suppresses pulmonary metastasis of osteosarcomain vivordquo International Journal of Cancer vol 104 no 2 pp 147ndash154 2003

[19] M Egeblad and Z Werb ldquoNew functions for the matrix met-alloproteinases in cancer progressionrdquo Nature Reviews Cancervol 2 no 3 pp 161ndash174 2002

[20] L A Liotta and E C Kohn ldquoThe microenvironment of thetumourmdashhost interfacerdquoNature vol 411 no 6835 pp 375ndash3792001

[21] K Podsypanina Y-C N Du M Jechlinger L J Beverly DHambardzumyan andH Varmus ldquoSeeding and propagation ofuntransformedmouse mammary cells in the lungrdquo Science vol321 no 5897 pp 1841ndash1844 2008

[22] F V Jacinto E Ballestar S Ropero and M Esteller ldquoDiscoveryof epigenetically silenced genes by methylated DNA immuno-precipitation in colon cancer cellsrdquo Cancer Research vol 67 no24 pp 11481ndash11486 2007

[23] J Taylor J Hickson T Lotan D S Yamada and C Rinker-Schaeffer ldquoUsing metastasis suppressor proteins to dissectinteractions among cancer cells and their microenvironmentrdquoCancer and Metastasis Reviews vol 27 no 1 pp 67ndash73 2008

[24] K Heese T Yamada H Akatsu et al ldquoCharacterizing thenew transcription regulator protein p60TRPrdquo Journal of CellularBiochemistry vol 91 no 5 pp 1030ndash1042 2004

[25] M Pancione L Sabatino A Fucci et al ldquoEpigenetic silencing ofperoxisome proliferator- activated receptor 120574 is a biomarker forcolorectal cancer progression and adverse patientsrsquo outcomerdquoPLoS ONE vol 5 no 12 Article ID e14229 2010

[26] B Bierie and H L Moses ldquoTumour microenvironmentmdashTGFΒ themolecular Jekyll andHyde of cancerrdquoNature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Sarcoma

manipulated cells that constitutively express the bacteriallacZ gene [5] Tracking the metastasizing tumor cells indistant tissues and organs by X-gal staining down to thesingle cell level demonstrated for the first time that Dunncells spontaneously metastasize from sc primary tumorsto lungs and livers with the same incidence as LM8 cellsHowever Dunn cells different from LM8 cells did not growto macrometastatic foci and remained in the lung and theliver as micrometastases consisting of small cell clusters orsingle cells until the end of the study on day 25 Thesemicrometastases remained undetectable with standard tissuestaining techniques such as hematoxylin amp eosin stainingThese findings explain why the Dunn cells were so farconsidered as nonmetastatic

Metastasis is a complex multistep process and multiplegenes and factors regulate individual steps [6ndash8] The dif-ferent cellular processes along the metastatic cascade arealso subject to variable regulation in time including periodsof metastatic latency [9] The results of the recent studythat compared the metastatic potencies and properties ofthe LM8 and parental Dunn cells in C3H mice during anexperimental period of 25 days raised the question whethertheDunn cells lacked cellularmechanisms needed for growthand development to macrometastases in the metastatic nicheor whether they go through a longer period of metastaticlatency than the LM8 cells upon arrival in the target organs

Here metastatic properties of Dunn and LM8 cells werefurther analyzed in vitro and two follow-up studies werecarried out in vivo to answer the question raised by therecent report [5] In a first in vivo time-course study LacZ-transduced Dunn and LM8 cells were intravenously (iv)injected into the tail vein of C3Hmice and individual animalsin both groups of mice were randomly selected and sacrificedat different time points up to 25 days after tumor cellinjection In a second survival study C3H mice iv injectedwith lacZ-transduced Dunn and LM8 cells were examineduntil they became moribund and had to be sacrificed Inthe time-course study the lung liver and kidney and inthe survival study additionally the urogenital tract and insome animals the spinal cords were analyzed for micro-and macrometastases by X-gal staining of respective organsand tissues upon sacrifice Interestingly Dunn cells formedmacrometastases in the lung and in the liver and kidneywith a delay of one and two weeks respectively compared toLM8 cells The delay in the development of macrometastaseswas also reflected by significantly longer median survival ofDunn- compared to LM8-cell-injected mice In conclusionDunn cells much like LM8 cells are equipped with all thecellular mechanisms needed for dissemination to differentorgans of C3H mice but different from LM8 cells Dunncells appear to go through a period ofmetastatic latency uponarrival in these organs

2 Materials and Methods

21 Cell Lines Themouse OS cell lines Dunn and LM8 wereretrovirally transduced with a lacZ gene as described [5] andcultured at 37∘C in DMEMHam F12 (1 1) medium (Invitro-gen Carlsbad CA) supplemented with 10 heat-inactivated

FCS in an incubator with a humidified atmosphere of 5CO2

22 RNAExtraction andMicroarray Analysis RNA isolationpreparation and array hybridization were performed asdescribed [10] Briefly total RNA isolated from Dunn andLM8 cells was quantified by measuring the absorption at260 and 280 nm and RNA integrity was evaluated by agarosegel-electrophoresis Complementary RNA preparation andarray hybridization were performed by the FunctionalGenomics Center (Zurich Switzerland) using AffymetrixMouse Genome 430 20 (45101 probe sets) arrays Raw datanormalization and statistical analysis were performed usingRACE (httpraceunilch)The obtained data sets were thenfiltered by setting a fold-change cutoff gt2 (both directions)and 119875 lt 005 Resulting data were then grouped into twosets one containing the upregulated genes in LM8 comparedto Dunn and the other containing the downregulated genesBoth sets of data were then analyzed for significant enrich-ment in certain Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways via the Database for Annotation Visu-alization and Integrated Discovery (DAVID) bioinformaticsresources [11]

23 cDNA Synthesis and Real-Time PCRAnalysis 1 120583g of totalRNA was reverse-transcribed to cDNA using high capacityRNA-to-cDNA kit (Applied Biosystems Foster City CAUSA) according to the protocol provided by the manu-facturer Three independent RNA extracts from the indi-vidual cell lines were reverse-transcribed in a final vol-ume of 10 120583L Real-time PCR was carried out in StepOne-Plus Real-Time PCR System (Applied Biosystems USA)in 96-well plates Primers for Mmp2 (FP CGCTCA-GATCCGTGGTGA RP CGCCAAATAAACCGGTCC-TT) Bhlhb9 (FP CCAGCCAGAGGGAAGAATAGC RPAAAGGCAGCAGAACACAAAGC) Fn1 (FP CGAAGC-CGGGAAGAGCAAG RP CGTTCCCACTGCTGATTT-ATCTG) Src (FP TACCACTCCTCAGCCTGGAT RPACACGAGGAAGGTGGATGTC) and Gapdh (FP TGC-AGTGGCAAAGTGGAGAT RP TTTGCCGTGAGTGGA-GTCATA) were designed with the NCBI Primer BLASTsoftware (httpwwwncbinlmnihgovtoolsprimer-blast)PCRs from individual RT reactions were carried out intriplicate cDNA equivalent to 50 ng of RNA and appropriateprimers were added to Power SYBR Green PCR MasterMix (Applied Biosystems USA) and the samples were pre-incubated at 50∘C for 2min and at 95∘C for 10min and thensubjected to 40 cycles of incubation at 95∘C for 15 s and at60∘C for 1min The threshold for Ct values was set to 0325To verify the amplification of a single product in any of thePCR reactions a melting curve was generated and analyzedafter every run Relative expression levels were calculated bythe comparative cycle threshold (ΔΔCT) method and werenormalized by GAPDH expression

24 Western Blotting Cells were grown to 80 confluence innormalmedium For supernatant (SN) preparation cells werestarved over night with 2mL of serum-freemediumThenext

Sarcoma 3








4 Sarcoma

3 Results








Sarcoma 5


unn

LM8

(a)

0 60 1200

50

100

DunnLM8

Time (min)

Part

icle

s (

tim

e zer

o) lowast lowast

lowast

(b)

Dunn LM800

05

10

15

20

25

Part

icle

s (gt1000

pixe

ls

)

lowast

(c)Dunn LM8

0

200

400

600

800

Wou

nd w

idth

(120583m

)

lowast

(d)Dunn LM8

00

02

04

06

Cell

scm

2(times10

6)

lowast

(e) (f) (g)

0 20 40 60 80 1000

10

20

30

LM8Dunn

Time (h)

Evas

ion

even

ts

lowast

lowast

(h)

Dunn LM80

5

10

15

20

Evas

ion

even

ts

lowast

(i)




6 Sarcoma

0

1

2

3

420

40

60

80

DunnLM8

Mmp2 Bhlhb9 Fn1 Src

2(minusΔΔ

Ct)


(a)

LM8

Dun

n

LM8

Dun

n

SN 2SN 1

MMP2

(b)

Dunn LM80

5

10

Fold

chan

ge

(c)






Sarcoma 7


Dunn

LM8

(a)

Dunn

LM8


(b)

Dunn

LM8


(c)

Figure 3 Continued

8 Sarcoma

First micromets in


First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)





4 Discussion



Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 3








4 Sarcoma

3 Results








Sarcoma 5


unn

LM8

(a)

0 60 1200

50

100

DunnLM8

Time (min)

Part

icle

s (

tim

e zer

o) lowast lowast

lowast

(b)

Dunn LM800

05

10

15

20

25

Part

icle

s (gt1000

pixe

ls

)

lowast

(c)Dunn LM8

0

200

400

600

800

Wou

nd w

idth

(120583m

)

lowast

(d)Dunn LM8

00

02

04

06

Cell

scm

2(times10

6)

lowast

(e) (f) (g)

0 20 40 60 80 1000

10

20

30

LM8Dunn

Time (h)

Evas

ion

even

ts

lowast

lowast

(h)

Dunn LM80

5

10

15

20

Evas

ion

even

ts

lowast

(i)




6 Sarcoma

0

1

2

3

420

40

60

80

DunnLM8

Mmp2 Bhlhb9 Fn1 Src

2(minusΔΔ

Ct)


(a)

LM8

Dun

n

LM8

Dun

n

SN 2SN 1

MMP2

(b)

Dunn LM80

5

10

Fold

chan

ge

(c)






Sarcoma 7


Dunn

LM8

(a)

Dunn

LM8


(b)

Dunn

LM8


(c)

Figure 3 Continued

8 Sarcoma

First micromets in


First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)





4 Discussion



Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Sarcoma

3 Results








Sarcoma 5


unn

LM8

(a)

0 60 1200

50

100

DunnLM8

Time (min)

Part

icle

s (

tim

e zer

o) lowast lowast

lowast

(b)

Dunn LM800

05

10

15

20

25

Part

icle

s (gt1000

pixe

ls

)

lowast

(c)Dunn LM8

0

200

400

600

800

Wou

nd w

idth

(120583m

)

lowast

(d)Dunn LM8

00

02

04

06

Cell

scm

2(times10

6)

lowast

(e) (f) (g)

0 20 40 60 80 1000

10

20

30

LM8Dunn

Time (h)

Evas

ion

even

ts

lowast

lowast

(h)

Dunn LM80

5

10

15

20

Evas

ion

even

ts

lowast

(i)




6 Sarcoma

0

1

2

3

420

40

60

80

DunnLM8

Mmp2 Bhlhb9 Fn1 Src

2(minusΔΔ

Ct)


(a)

LM8

Dun

n

LM8

Dun

n

SN 2SN 1

MMP2

(b)

Dunn LM80

5

10

Fold

chan

ge

(c)






Sarcoma 7


Dunn

LM8

(a)

Dunn

LM8


(b)

Dunn

LM8


(c)

Figure 3 Continued

8 Sarcoma

First micromets in


First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)





4 Discussion



Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 5


unn

LM8

(a)

0 60 1200

50

100

DunnLM8

Time (min)

Part

icle

s (

tim

e zer

o) lowast lowast

lowast

(b)

Dunn LM800

05

10

15

20

25

Part

icle

s (gt1000

pixe

ls

)

lowast

(c)Dunn LM8

0

200

400

600

800

Wou

nd w

idth

(120583m

)

lowast

(d)Dunn LM8

00

02

04

06

Cell

scm

2(times10

6)

lowast

(e) (f) (g)

0 20 40 60 80 1000

10

20

30

LM8Dunn

Time (h)

Evas

ion

even

ts

lowast

lowast

(h)

Dunn LM80

5

10

15

20

Evas

ion

even

ts

lowast

(i)




6 Sarcoma

0

1

2

3

420

40

60

80

DunnLM8

Mmp2 Bhlhb9 Fn1 Src

2(minusΔΔ

Ct)


(a)

LM8

Dun

n

LM8

Dun

n

SN 2SN 1

MMP2

(b)

Dunn LM80

5

10

Fold

chan

ge

(c)






Sarcoma 7


Dunn

LM8

(a)

Dunn

LM8


(b)

Dunn

LM8


(c)

Figure 3 Continued

8 Sarcoma

First micromets in


First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)





4 Discussion



Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Sarcoma

0

1

2

3

420

40

60

80

DunnLM8

Mmp2 Bhlhb9 Fn1 Src

2(minusΔΔ

Ct)


(a)

LM8

Dun

n

LM8

Dun

n

SN 2SN 1

MMP2

(b)

Dunn LM80

5

10

Fold

chan

ge

(c)






Sarcoma 7


Dunn

LM8

(a)

Dunn

LM8


(b)

Dunn

LM8


(c)

Figure 3 Continued

8 Sarcoma

First micromets in


First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)





4 Discussion



Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 7


Dunn

LM8

(a)

Dunn

LM8


(b)

Dunn

LM8


(c)

Figure 3 Continued

8 Sarcoma

First micromets in


First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)





4 Discussion



Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 Sarcoma

First micromets in


First micromets in


First micromets in


First macromets

in lung

First macromets

in liver and kidney


Dunn

LM8

(d)





4 Discussion



Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 9






































10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















10 Sarcoma














































Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 11

0 50 1000

50

100

Dunn

LM8

Time (days)

Surv

ival

rate

()

(a)

Dunn LM8

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)




12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















12 Sarcoma








Acknowledgments



References


















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 13
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article reduced latency in the metastatic niche...

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