supplementary data supplementary methods...histopathology and immunohistochemistry for hematoxylin...

SupplementaryDataSupplementaryMethodsRNA-Seqworkflow

Initial quality control analysis of RNA-Seqdata for each samplewas performedusing FastQC

(version0.11.2).EachsamplewasalignedusingtheTophataligner(version2.0.13)(24).Samtools

software(version1.0_BCFTools_HTSLib)wasusedtosortandindexthebamfiles(25).Cuffquant

(Cufflinksversion2.2.1)wasusedtogeneratetranscriptabundancefiles(optionsusedinclude

themulti-read-correct,max-bundle-frags<10000000>andmask-file(forgenes<200bp).Once

all sampleswithineachspeciesweremappedandabundanceestimate fileswerecompleted,

Cuffnorm(Cufflinksversion2.2.1),wasusedtogenerateatableofFragmentsPerKilobaseOf

ExonPerMillionFragmentsMapped(FPKM)valuesforgeneswithineachsample(26).Cuffquant

wassettomaskgenes<200bp.TominimizetheeffectsofdividingFPKMvaluesbynumbersclose

to0andstochasticnoise,0.1wasaddedtoeachFPKMvalue(27,28).TheFPKMfilesareavailable

atGEOGSE87686.MappingstatisticsaresummarizedinSupplementalTable3.

Referencegenomes

AspartoftheRNA-Seqworkflow,genesweredefinedbythegenometranscriptfilesfor

eachspecies.TheUniversityofCalifornia,SantaCruz(UCSC)GenomeBrowserversionhg19

(GRCh37assembly)ofthehumanreferencegenomeandEnsemblGenesv70wereusedfor

mappinghumansequence.VersioncanFam3(BroadInstitutev3.1)wasusedasthedog

referencegenomeinthisstudy.TheBroadInstituteprovided.bedfiles(personal

communication)andfromthesea.gtffilewascreated.UCSCversionmm10(GRCm38,Genome

ReferenceConsortiumMouseBuild38(GCA_000001635.2))wasusedasthemousereference

genome.

Correlationanalyses

PairwisePearsoncorrelationcoefficientswerecalculatedusing12,062genescommon

betweenhumanOS(HOS1,HOS2,HOS3),dogOS(DOS),MouseOS(MOS),andTCGAdatafor

other human cancers. Twenty-five samples from RNA-Seq expression data were randomly

selected for TCGA primary tumors from Cervical Squamous Cell Carcinoma and Endocervical

Adenocarcinoma (CESC), ColonAdenocarcinoma (COAD),Glioblastoma (GBM),AcuteMyeloid

Leukemia(LAML),Prostate(PRAD),andThyroid(THCA)cancerdatasets.

Transcriptomeanalyses

OptiType,aprecisionHLAtypingtool(29)wasusedtoidentifyhumanOSsamplesderived

from the same patient. In the 44 human OS samples, five pairs ofmatching human patient

sampleswereidentifiedusingOptiType.For4ofthe5pairs,theprovidedage,sex,andraceof

thepatientswerealsoidentical(the5thpairdidnothavecompletemetadatainformation).These

duplicatesampleswereallderivedfromtheUMNBioNETrepository,andnoothermatchingpairs

wereobservedacrossallOShumansequencingdatausedinthisstudy.

HistopathologyandImmunohistochemistry

For hematoxylin and eosin staining, FFPE sections (4 µm) were de-paraffinized and

rehydratedusingstandardmethods.SlideswereplacedintoHarrishematoxylinfor5minutes.

Slideswererinsed,dippedtwiceinacidalcohol,rinsedandplacedintoammoniawater(bluing)

for2minutes followedbya15minute tapwater rinse.After the tapwater rinseslideswere

placedin80%ethanolfor2minutesandintoEosinYfor1minute.Aftertheeosinslideswererun

throughgradedalcohols,dehydratedandcover-slipped.Forimmunohistochemistry,sections(4

µm)werede-paraffinizedandrehydratedusingstandardmethods.Antigenretrievalwasdone

byplacing slides in a steamer, in 6.0 pHbuffer (RevealDecloaking reagent, BiocareMedical,

Concord,CA)for30minat95-98°C,followedbya20mincooldownperiodforallantibodies

except Calprotectin (MAC387), where antigen retrieval was not needed. Slides were then

incubatedwithProteinaseKfor5minutesat25°C,rinsedandplaceintoTris-bufferedsalinewith

Tween-20 (TBST). Subsequent stepswere automated using an immunohistochemical staining

platform(Nemesis,Biocare).Endogenousperoxidaseactivitywasquenchedbyslideimmersion

in3%hydrogenperoxidesolution(Peroxidazed,Biocare)for10minfollowedbyTBSTrinse.A

serum-freeblockingsolution(BackgroundPunisher,Biocare)Medical,Concord,CA)wasplaced

on sections for10min.Blocking solutionwas removedand slideswere incubated inprimary

antibody diluted in 10% blocking solution/90% TBST.Mousemonoclonal anti-Vimentin(clone

V9)(Zymed;1:400), rabbit monoclonal anti-CD3(clone SP7)(Thermo Scientific, Kalamazoo, MI

1:400), rabbit monoclonal anti-Calprotectin(clone

MAC387)(Invitrogen,Rockford,IL;1:100&1:200),wereincubatedfor60minatroomtemperature

followedbyTBSTrinseanddetectionwithNovocastraNovolinkPolymerKit(LeicaMicrosystems

Inc.,BuffaloGrove, IL)usingthemanufacturer’sspecifications.Allslidesthenproceededwith

TBST rinse and detectionwith diaminobenzidine (DAB) (Covance, Dedham,MA). Slideswere

incubatedfor5minfollowedbyTBSrinsethencounterstainedwithCATHematoxylin(Biocare,

Concord,CA)for5min.Slideswerethendehydratedandcover-slipped.

Statistics

Statistical significancewas calculated using the log-rank test or by Fisher’s exact test

dependingonanalysisandap<0.05wasconsideredsignificant.Kaplan-Meier(KM)survivalplots

weregeneratedusingthe‘survival’packageinR(Version0.98.1103)(30,31).TheGCESSvalues

wereusedtorankthetumorsintoquartilegroupsandthequartilegroupsweresystematically

testedforassociationwithoutcome.Numberoftumorswithoutcomeinformation,thenumber

ofoutcomeeventsused inall statisticalanalysesandquartile cut-off values foreverycluster

analyzedinthispaperareprovidedasSupplementalTable2.Samplesizewasbasedonavailable

data.

OutcomeDefinitionsandMeasurement

Toidentifyassociationsbetweentranscriptionalvariationandoutcomes,severaldifferent

typesofoutcomedatawereutilized.Forthecaninedatasetsurvivaltimeposttumorresection

wasavailablefor19tumors(averagetimeuntildeath137days).FortheHumanHOS2dataset,

followupdatawasavailablefor35tumors,(averagefollowuptime733days)andduringthis

time17deathsoccurred.Additionally,theobservationofpresenceofmetastasesatdiagnosis

wasalsoavailablefor17patients.ForGSE21257,followupdatawasavailablefor53patients

withanaveragefollowuptimeof2056days.23deatheventswereobservedandmetastatic

diseasewasobservedin34ofthesepatients.

SupplementalFigure1.Realhumandataclustered

SupplementalFigure2.Randomhumandataclustered

SupplementalFigure3.Permutedhumandataclustered

SupplementalFigure4.Realmousedataclustered

SupplementalFigure5.Randommousedataclustered

SupplementalFigure6.Permutedmousedataclustered

SupplementalFigure7.Realdogdataclustered

SupplementalFigure8.Randomdogdataclustered

SupplementalFigure9.Permuteddogdataclustered

SupplementalFigure10.Examplesof4immunohistochemistrygroups

Supplemental Figure 11. KM significance in real, random, and permuted human and dog

datasets.

SupplementalFigure12.KMhumancluster-1



SupplementalFigure15.KMdogcluster-3

SupplementalFigure16.GSE212257Arraycluster-3associationwithOutcome

SupplementalFigure17.GSE212257Arraycluster-5associationwithOutcome

SupplementalFigure18.GSE212257Arraycluster-3associationwithMetastasis



SupplementalFigure21.CellCycleGCESSplottedagainstImmune-1andImmune-2GCESS

SupplementalFigure22.Modeldepictinghowimmunecellcomponentsmaybepreventingthe

occurrenceofmetastasisinOSpatients.

SupplementalFigure23.FlowchartofOSsamplesutilizedinthiswork

SupplementalTable1.Samplemetadatatable.

Tab1HumanTab2DogTab3MouseTab4Humanoutcome-survivaltime,presenceofmetastasisatdiagnosisTab5Dogoutcome-survivaltimeTab6GSE21257outcome-survivaltime,metastasistimeSupplementalTable2.TumorCounts,numberofOutcomeeventsandGCESSOutcome

thresholdvalues

SupplementalTable3.

ReadCounts,mappingpercentageandexpressedgenecountforallRNAseqsamples.Tab1HumanTab2DogTab3Mouse

SupplementalTable4.Genelistsidentifiedintheclustering.Tab1GenesinHumanclusters(n=7),mouseclusters(n=11),dogclusters(n=5),GSE212257(n=14)Tab2OverlapcountsofRNASEQclusterlists.Tab3Overlapofhumancluster1andhumancluster8withLM22signature

SupplementalTable5.IPAenrichmentanalysesofrecurrenthumangeneclusters.

SupplementalTable6.GeneClusterExpressionSummaryScores.

Tab1HumanGCESSvaluesTab2MouseGCESSvaluesTab3DogGCESSvaluesTab4GSE212257GCESSvalues

SupplementalTable7.ImmunohistochemistryvalidationofImmune1andImmune2GCESS

SupplementalFigure1RealHumandata

SupplementalFigure2RandomHumandata

SupplementalFigure3PermutedHumanData

SupplementalFigure4RealMouseData

SupplementalFigure5RandomMousedata

SupplementalFigure6PermutedMousedata

SupplementalFigure7RealDogdata

SupplementalFigure8RandomDogData

SupplementalFigure9PermutedDogData

SupplementalFigure10Examplesof4ImmunohistochemistryGroups

SupplementalFigure11A.

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival

Median survival= 540Median survival= NA

p = 0.00588

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q12 vs Q34

Time (days)

Surv

ival

Median survival= 690Median survival= 1260

p = 0.15

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.886

SupplementalFigure12HumanCluster1“Immune-1”associationwithOutcome

SupplementalFigure13HumanCluster4“CellCycle”associationwithOutcome

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival


p = 0.861

0 500 1500 2500 35000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.751

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.0577

SupplementalFigure14HumanCluster8“Immune-2”associationwithOutcome

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival


p = 0.14

0 500 1500 2500 35000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.261

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.0927

SupplementalFigure15CanineCluster3“CellCycle”associationwithOutcome

0 50 100 150 200 250 300

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival

Median survival= 165.6986301Median survival= 134.630137

p = 0.685

0 50 100 150 200 250 3000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.00313

0 50 100 150 200 250 300

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.00328

SupplementalFigure16GSE212257ArrayCluster3“Immune-1”associationwithOutcome

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival


p = 0.25

0 500 1500 2500 35000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.523

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.0335

SupplementalFigure17GSE212257ArrayCluster5“Immune-2”associationwithOutcome

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival


p = 0.233

0 500 1500 2500 35000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.0121

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.159

SupplementalFigure18GSE212257ArrayCluster3“Immune-1”associationwithMetastasis

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival


p = 0.00114

0 500 1500 2500 35000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.0558

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.000742

SupplementalFigure19GSE212257ArrayCluster5“Immune-2”associationwithMetastasis

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival


p = 0.000679

0 500 1500 2500 35000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.000131

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.00613

SupplementalFigure20GSE212257ArrayCluster7 “CellCycle”associationwithMetastasis

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q1 vs Q234

Time (days)

Surv

ival


p = 0.2

0 500 1500 2500 35000.

00.

20.

40.

60.

81.

0

Q12 vs Q34

Time (days)

Surv

ival


p = 0.0226

0 500 1500 2500 3500

0.0

0.2

0.4

0.6

0.8

1.0

Q123 vs Q4

Time (days)

Surv

ival


p = 0.0583

SupplementalFigure21HumanCellCycleGCESSplottedagainstImmune-1andImmune-2GCESS

WorseOutcome BetterOutcome

SupplementalFigure22

Follow up data

Samples Cohorts Species Outcome Analysis

Fig 3, KM analyses

Fig 4, KM analyses

Fig 3, KM analyses

Human

HOS1

Tissue (n= 46)

Bone Tissue (n=3)

Cells (n = 5)

HOS22 Tissue (n=35)Time to Death

Metastases at Diagnosis

HOS33 Tissue (n=25)

GSE212574 Tissue (n=53)

Time to Death

Time to Metastases

Mouse

Tissue (n=92)

Cells (n=11)

Dog

Tissue (n=31) Time to Death

Cells (n=2)

Osteoblast Cells (n=1)

MOS1

DOS1

SupplementalFigure23FlowchartofOSsamplesutilizedinthiswork

Figure

Fig 1, CorrelationFig 2, ClusteringFig 3, GCESS



Fig 4, Clustering



1. RNA-Seq dataproducedforthisstudy.2. RNA-Seq datadownloadedasdescribedinthemethodssection.3. RNA-Seq datapreviouslygeneratedasdescribed.4. Arrray datadownloadedasdescribedinthemethodssection.

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