Ovarian Cancer Derived Exosomal Fibronectin Induces Pro Inflammatory Il 1beta

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Exosomes and Microvesicles

Ovarian Cancer-Derived ExosomalFibronectin Induces Pro-Inflammatory IL-1Original Research Article

Safinur Atay1, Carolyn D. Roberson2, Cicek Gercel-Taylor3 and Douglas D. Taylor3,*

1 Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA2 Department of Microbiology & Immunology, University of Louisville School of Medicine, Louisville, KY, USA3 Department of Obstetrics, Gynecology & Womens Health, University of Louisville School of Medicine, Louisville, KY, USA* Corresponding author E-mail: [email protected]

Accepted 19 Feb 2013

2013 Atay et al.; licensee InTech. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract The tumour microenvironment is

characterizedbyproinflammatoryprofiles, including

interleukin1 (IL1). This profile is generated by

infiltrating macrophages following interactions with

tumours or their components. The objectives of this

study were to identify whether tumour exosomes

could induce macrophage IL1 production and the

mechanism involved. Exosomes were isolated from

ovarian cancer patients and ovarian tumour cellsby

chromatography and ultracentrifugation. Specific

exosomalproteinsweredefinedbymassspectrometry

(MS) and confirmed by Western immunoblotting.

UsingmacrophagelikeTHP1cells,inductionofIL1

releasewasinvestigated byELISA.RGDpeptideswere

used to block fibronectin binding by THP1 51

integrin. Exosomes isolated from ovarian cancer

patients and from ovarian cancer cells were

demonstrated, byMSand immunoblotting, toexpress

fibronectin. Incubation of THP1 cells with these

exosomes induced proinflammatory cytokines, in

particular IL1. Blocking of THP1 binding of

exosomal fibronectin with RGD peptides abrogated

exosomemediatedIL1 productionanddownstream

phosphorylation of Akt and cJun. Although cancerpatientsgenerallyexhibitincreasedlevelsofIL1,the

underlying mechanism is unclear. Here, tumour

derived exosomes are demonstrated to induce pro

inflammatory cytokine in macrophages including IL

1,whoseinductionismediatedbyfibronectin.

KeywordsExosomes,Fibronectin,IL1,Macrophages

1.Introduction

Macrophageshavebeen implicated inphagocytosisand

tissue remodelling and production of cytokines and

chemokines [1, 2]. Unlike many cells of the innate

immune system, macrophages exhibit a high degree of

plasticityandcandifferentiatedependingon the signals

in their environment [3]. Based on their receptors and

cytokineproduction,macrophagescanbedefinedasM1

orM2macrophages.ProinflammatoryM1,orclassically

activated, macrophages exhibit upregulation of pro

inflammatorycytokinesandchemokines,includingTNF

, IL12, IL6,CCL2and IL1, in addition to increased

production of reactive oxygen species and nitrogen

intermediates [4]. In contrast, antiinflammatory M2, or

alternatively activated, macrophages are polarized bystimuli such as IL4, IL13, IL10 or glucocorticoid

1Safinur Atay, Carolyn D. Roberson, Cicek Gercel-Taylor and Douglas D. Taylor:

Ovarian Cancer-derived Exosomal Fibronectin Induces Pro-inflammatory IL-1

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hormones [4] and upregulate scavenger,mannose and

galactose receptors. They are IL1 receptor antagonists

and downregulate IL1 and other proinflammatory

cytokines[5].

Themalignantbehavioursoftumourcellsaredefinedbythemicroenvironment,wheremacrophagesrepresentthe

major population of immunologic cells inltrating the

tumours.These infiltratingmacrophages are referred to

as tumourassociated macrophages (TAMs). While

initiallyitwasbelievedthatthesemacrophagesexhibited

antitumour activity, the inltration of TAM into

tumours, such asbreast, ovarian, prostate and cervical

cancers, has been correlated with poor prognosis and

shortsurvival[6].Thisadversefunctionofmacrophages

hasbeen attributed to their immunosuppressive nature

[7]. Recent studies have demonstrated that TAMs

promote tumour cell proliferation, invasion andmetastasis,aswellasangiogenesis,whichisessentialfor

tumourgrowth [8].TAMssecreteavarietyofcytokines,

growth factors and enzymes, which promote tumour

metastasisandangiogenesis,suchasvascularendothelial

growth factor, IL8, epidermal growth factor, platelet

derived growth factor, tumour necrosis factor (TNF)

andmatrixmetalloproteinases[9,10].

Cytokines coordinate the immune response and are

released in response to infection, injury, stress or

trauma. Cytokines regulate differentiation and

activation of various immune cells and coordinateinflammatoryreactions,suchas the inductionofacute

phase proteins. The hyperactivation of pro

inflammatorycytokinesand/or the impairmentofanti

inflammatory cytokines can lead to pathological

conditions, such as allergic reactions, sepsis and

dysplasia. IL1, TNF and IL17 are prominent pro

inflammatory cytokines,whereas IL10 and TGF are

characterizedbyantiinflammatoryproperties.Thepro

inflammatoryphase is characterizedby theproduction

ofproinflammatorycytokines,includinginterleukin1

(IL1) [11]. IL1 is a potent multifunctional pro

inflammatory cytokine produced by monocytes and

tissue macrophages. IL1 promotes multiple events,

such as attraction and activation of immune cells,

stimulation of endothelial cell adhesion molecule

expression, and enhanced expression of extracellular

matricesandmatrixdegradingenzymes.

Our previous work has demonstrated that exosomes

derived from a first trimester extravillous trophoblast

were capable of inducing themigration ofmonocytes

and educating these cells to produce a pro

inflammatorycytokineprofile, including IL1 [12]. IL

1 inductionby exosomeswasmediatedby a ligand

receptor interaction and was shown not to requireexosome uptake. Other studies have shown that

interactions between components of the extracellular

matrixandmacrophages lead to theproduction inpro

inflammatory cytokines [13].Proteins shown to induce

proinflammatory cytokines by macrophages include

Thy1, vitronectin, integrins, thrombospondin,myosin

and fibronectin. Fibronectin is present in theextracellularmatrix ofmost tissues,where it supports

basic cellular processes including cell adhesion,

migration, survival and growth. Fibronectin is a

modular protein composed of independently folded

domains(typesI,IIandIII),whichrepresentregionsof

amino acid sequence homology [14]. Within the

extracellularmatrix,fibronectinispolymerizedandcan

be subjected to tensional forces generatedby the cells

that expose cryptic activities within the molecule,

includingbindingsitesforintegrinsandgrowthfactors

[15]. In general, fibronectins are a class of large

glycoproteins with known adhesive activity forcollagen, fibrin, heparin and cell surfaces and are

associatedwithwoundhealing,plateletaggregationand

clotting, and cancermetastasis [16].A specific function

in cancerbiologyhasnotbeendefined for fibronectin,

due primarily to its ubiquitous expression in adult

human tissuesandplasma.Matsuuraetal. identifieda

group of fibronectin isoforms, containing anOlinked,

Nacetylgalactosaminylated hexapeptide within the

alternatively spliced type III connecting segment

[17].These unique fibronectin iosforms have been

identified within several human tumours and

developmentaltissues.Theseisoformsaredesignatedasoncofetalfibronectinandtheyappeartobedistinctfrom

plasmaandcellularfibronectinsbasedonmolecularsize

andglycosylationpatterns[18].

Inthepresentstudy,wedemonstratetheinductionofIL

1 by cancerderived exosomes and investigate the

mechanism underlying this induction. The study

addressedtheexpressionofexosomalcomponentslinked

with the induction of proinflammatory cytokines by

macrophages.Based onMS identification of fibronectin

on tumourderivedexosomes, thestudy investigated the

blockage of the exosomemediated events by RGD

analoguesandthemolecularmechanismlinkedwith the

exosomeassociated component(s) responsible for the

inductionofIL1.

2.MaterialsandMethods

2.1CellcultureconditionsTheULOcell linewasestablished from theascitesofa

patientwithStageIIIpapillaryserousadenocarcinomaof

the ovary.ULO cellsweremaintained in 75cm2 tissue

culture flasks in 20mlHyclone Roswell ParkMemorial

Institute (RPMI) 1640 culture media (ThermoScientific)supplemented with 2mM Lglutamine, 10% exosome

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depleted foetal bovine serum (FBS), 1mM sodium

pyruvate, 0.1mM nonessential amino acids and

100units/mL penicillinstreptomycin in a humidified

incubatorat37Cwith5%CO2.FBSassociatedexosomes

were removed by ultracentrifugation for 15 hours at

100,000xg.The resulting supernatantwas sterile filteredusinga0.22m filter.THP1acutemonocytic leukaemia

cells were obtained from ATCC (Rockville,MD, USA)

andweremaintained inRPMI 1640 supplementedwith

2mM Lglutamine, 10% FBS, 1mM sodium pyruvate,

0.1mM nonessential amino acids, 0.05mM 2

mercaptoethanoland100units/mLpenicillinstreptomycin

inahumidifiedincubatorat37Cwith5%CO2.

2.2Exosomeisolationfromculturemediaandpatientascites

To isolate tumour exosomes from culturemedia,ULO

cellswereutilized.ULOcellswereculturedforthree tofourdays(approximately85%confluentandgreaterthan

95% viability based on trypan blue exclusion). The

conditionedmedium was centrifuged at 400xg for ten

minutes toremovewholecellsand thissupernatantwas

then centrifuged at 15,000xg for 20minutes to remove

largemicrovesiclesandapoptoticvesicles.The resulting

cell free medium was concentrated by ultrafiltration

usinganAmiconstirredcellModel8200withamolecular

weight cutoffmembrane of 500,000Daltons (Millipore,

Billerica, MA). This concentrated material was then

chromatographically separated using a Sepharose 2B

column (2.5x30cm). The void volume fractions werepooled and centrifugedusingaTi90 rotor (Beckman) at

100,000 x g for one hour at 4C. The pellet was

resuspended in PBS and the vesicular protein

concentration determined using the DC protein assay

(BioRad Laboratories, Hercules, CA) according to

manufacturers instructions. This vesicle preparation

fromULOcellswastermedOEX.

Toisolateexosomesfrompatientascites,theascitesfluids

werecentrifugedat400xgfortenminutestoremovecells

and the supernatantwas centrifuged at 15,000xg for 20

minutestoremovecelldebrisandapoptoticvesicles.The

resultingsupernatantwasconcentratedbyultrafiltration

usinganAmiconstirredcellwithamolecularweightcut

off membrane of 500,000 Daltons (Millipore). This

concentrated material was then chromatographically

separatedusingaSepharose2Bcolumn (2.5x30cm).The

void volume fractions were pooled and centrifuged at

100,000 x g for one hour at 4C. The pellet was

resuspended in PBS and the protein concentration

determinedusing theDCprotein assay.To exclude the

possibility of endotoxin contamination in the exosome

preparations for both the culture and ascitesderived

vesicles, a LAL assay (Genscript, Piscataway, NJ) was

performed to quantify any endotoxin in the vesiclepreparations.Fortheexosomepreparationsusedinthese

studies, endotoxin concentrations were


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The diameter of the exosomeswere obtained from the

applicationoftheStokesEinsteinequations[20].

2.5Massspectrometryandproteomicanalysisof

ascitesderivedexosomes

Specific proteinbands from SDSPAGE gels of ascites

derived exosomalproteinswere reduced, alkylated and

trypsinized at 37C overnight with shaking. Briefly,

tryptic peptides were loaded onto a 1D capillary

chromatographycolumncomprisedofaneedletip(100x

365m fused silica capillary with an integrated, laser

pulled emitter tip) packed with 10cm of Jupiter 5m

RP300A (Phenomenex, Torrence, CA). Peptides were

eluted, ionized and sprayed into themass spectrometer

usinga120minutegradientfrom7%acetonitrile to80%

acetronitrile (plus 0.1% formic acid) at a flow rate of

200nL/min. Spectrawere acquiredwith aLTQOrbitrapXL Linear Ion TrapMass Spectrometer (Thermo Fisher

Scientific, Waltham, MA). During LCMS/MS analysis,

the mass spectrometer performed datadependent

acquisitionwithafullMSscanbetween300and2000m/z

followedbyMS/MS scans (35% collisionenergy)on the

sixmost intense ions from theprecedingMS scan.Data

acquisitionwasperformedusingdynamicexclusionwith

a repeat count of a one and three minute exclusion

durationwindow.

The acquired mass spectrometry data were searched

against a translated human genome database (HumanRefSeqXPs)usingtheSEQUESTalgorithmassumingfixed

modification of cysteine (+57 for carabmidomethylation)

andvariableoxidationofmethionine(+16 tomethionine).

Database analysis was performed with SequestSorcerer

(SageNResearch,SanJose,CA).Highprobabilitypeptide

and protein identifications were assigned from the

SEQUEST results using the ProteinProphet

(http://tools.proteomecenter.org/software.php) and SageN

Sorcerer statistical platforms. These data were loaded

onto Scaffold 3 proteomic analysis software to

qualitatively and quantitatively compare individual

LCMS analyses and provide approaches to functional

annotationofdata.

2.6Cytokineantibodyarray

To define the effects of tumourderived exosomes on

macrophage chemokine and cytokine profiles, their

productionbyTHP1cellswasquantifiedwithduplicate

arrays, each having duplicate spots for each cytokine

using Proteome profiler Human Cytokine Antibody

ArrayPanelAArrays(R&DSystems,Minneapolis,MN)

according to the manufacturers instructions. THP1

monocytic cellswere incubatedwith 100g/ml tumour

derived exosomes orwere untreated for 20 hours. Thecytokine arraymembraneswere incubatedwith 1ml of

conditioned media from each sample, diluted 1:3 and

15lofCytokineArrayPanelAdetectionantibodyat4C

overnight.Themembraneswerethenwashedthreetimes

with 20ml of 1 wash buffer and incubated with

horseradish peroxidaseconjugated streptavidin (1:2000

dilution).After30minutes,themembraneswerewashedthoroughly and exposed to a chemiluminescent

peroxidase substrate for fiveminutes in thedarkbefore

imaging. Membranes were exposed to xray film

(ResearchProductsInternational,MtProspect,IL).Asper

the manufacturers package insert, the cytokine array

data on developed Xray film was quantitated by

scanning the film on a transmissionmode scanner and

analysing the array image file using image analysis

software,UnScanit gel digitizing software version 6.1

(SilkScientificCorporation,Orem,UT).Positivecontrols

atthreespotswereusedtoidentifymembraneorientation

and tonormalize the results fromdifferentmembranes.Foreachspot,thespecificpixel levelwasdeterminedby

subtracting thebackground pixels from the total raw

pixel levels. To quantify relative change in cytokine

levels between samples, the average background

subtracted mean spot pixel densities of the pair of

duplicate spots representing each cytokine was

determined for each condition. To facilitate further

analyses, all spots in the arrays were quantified and

their specific intensity values were obtained by

subtracting thebackground intensity.Only differences

in cytokine levels that were twofold compared to

controlswereconsideredsignificant.

2.7ELISAdeterminationofIL1release

For ELISA determination of IL1 human THP1, cells

(1x106 cells/well) were incubated with exosomes at

100g/mlforsixhours.FortheELISAanalysis,1mMATP

wasaddedfor30minutestoinducematureIL1release.

Culture supernatants for the treatedhumanTHP1 cells

wereharvestedandtheIL1levelsweremeasuredusing

IL1ELISAkit (eBiosciences,SanDiego,CA)according

tothemanufacturersinstructions.

2.8Co

Culture

of

THP

1monocytes

with

integrin

functioninhibitorypeptides

ToassessIL1production,THP1cells(5x106cells/well)

were cultured in RPMI 1640 supplemented media (as

previouslydescribed) alone, coculturedwith 100g/mL

ULO culturederived exosomes (OEX), or cocultured

with 100g/mL ULO culturederived exosomes (OEX)

plus peptide mimics of fibronectin binding (Sigma

Aldrich, St. Louis, MO) at 0.5mg/mL for 30 minutes.

Peptidemimicsinclude:anegativecontrolpeptide(Gly

ArgAlaAspSerProLys) that does not block integrin

binding (designatedasCP),anantagonistpeptide (GlyArgGlyAspSer) of integrin function (designated as



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ANT) and an inhibitorypeptide (GlyArgGlyAspThr

Pro) of fibronectin, type 1 collagen and vitronectin

(designated BFCV) [15]. Cultures were incubated with

OEX for sixhourswithorwithout theadditionof1mM

ATPfor30minutesat37C.Postincubation,supernatants

of cultures were harvested and subjected to ELISA

analysis using a commercially available IL1 kit

(eBiosciences,SanDiego,CA)todeterminetheamountof

IL1released.

2.9KineticsofTHP1,OEXandintegrinfunction

inhibitorypeptidecocultures

THP1cellswereunstimulated,stimulatedwithOEX,or

stimulated with OEX + peptide mimics of fibronectin

binding (aspreviouslydescribed)for0,30,60,120,or240

minutes. Total intracellular protein extracts were

preparedbyadding100LofRIPAbufferplusproteaseand phosphatase inhibitors/106 cells (ThermoScientific).

The extractswere incubated on ice for 30minutes and

centrifuged at 12,000rpm for ten minutes. Protein

concentration was determined using the DC Protein

Assay(BioRadLaboratories).Cellularprotein(30g)was

added to Laemmli reducingbuffer andboiled for five

minutes. Sampleswere electrophoresed at 100V for 1.5

hours and the resulting gel was transferred to

nitrocellulosemembranes (BioRad Laboratories) at 98V

for onehour on ice.Membraneswereblockedwith 5%

nonfatdrymilkin1xTBS+0.05%Tween20(1xTBST)for

onehourandincubatedonarockerovernightat4Cwithanti pAkt2 (clone B5), anti pser73 cJun and antiactin

(clone C4). All antibodies are from Santa Cruz

Biotechnology.Afteranovernight incubation,membranes

werewashed three timeswith 1xTBST and incubated at

roomtemperatureforonehourwithHRPconjugatedanti

mouse or antirabbit IgG (BioRad Laboratories).

Subsequently,membranesweredevelopedusing Immun

StarHRPChemiluminescentKit(BioRadLaboratories)and

visualized on Biomax MR film (Kodak). Western blot

images for pAkt2, pSer73 cJun and actinwere digitized

andrelativechangeinexpressionofproteinincomparison

to the control was quantified. Statistical analyses were

performedusingthettestandcalculatedusingGraphPad

Prism5.01graphingandstatisticalsoftware.Apvalueof


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Figure2.Mass spectrometricdeterminationof specificproteinsassociatedwithpatientascitesderivedvesicles.PanelApresentstheImperialpurplestained10%SDSPAGEofvesicularproteinsfromULL andULTB. Panel B shows themass spectrometrydefined proteins from bands one, two and three with the

correspondingpeptidecountandionscore.

Since previous studies have demonstrated the role of

tumourcellassociatedfibronectin intheinductionofIL

1,thepresenceoffibronectinwasconfirmedonvesicles

isolated in vitro from conditionedmedia ofULO cells

(OEX,Figure3A)andinvivofromovariancancerpatients

(ULL, ULO and ULTB, as well as normal control,

Figure3B).These resultsconfirm theenhancedpresence

of fibronectin on these tumourderived vesicles. In

addition,specificmarkerswereevaluatedbetweenULO

cells and their corresponding vesicles (OEX, Figure 3A)

using Western immunoblotting. These results also

demonstrated the enriched presence of CD63 on the

vesicles,establishingtheiridentityasexosomes.

Figure3.Western immunoblot identificationof specificproteinmarkersoncellsandcorrespondingextracellularvesicles.Panel

ApresentsmarkersassociatedwithULOcellsandextracellularvesiclesreleasedbyULOcells(OEX).PanelBpresentspresence

of fibronectinwith extracellular vesicles isolated from normal

humanABserm(normal)andthreeovariancancerpatients.

3.3ExosomeinductionofIL1releasebyhumanmacrophages

Since thepresenceof tumourshasbeen linkedwith the

inductionofaproinflammatorymicroenvironment.The

initialquestionwaswhether exosomesderived from an

ovariantumourwouldinducetheTHP1cellstoproduce

aproinflammatoryprofile.Using theProteomeprofiler

Human Cytokine Antibody Array Panel A Array, theinduction ofmultiple cytokines/chemokinesby tumour

derived OEX was evaluated (Figure 4). When THP1

monocytic cellswere incubatedwith 100g/ml tumour

derived exosomes, 14 of the 36 cytokines/chemokines

were inducedbygreater than twofold.Of these,GRO,

IL1,IL1,IL8,IL23,MCP1(CCL2),MIP1andTNF

were inducedbymore than 20fold (versus the controltreated).OfparticularinterestwasthatIL1wasinduced

by 350fold following exposure to tumourderived

exosomes.

Based on this major induction of IL1, the exosome

component that might mediate this pathway was

evaluated. Based on our previous mass spectrometric

evaluationofexosomalcomponents,thepotentialroleof

fibronectin in this exosomeinduction was analysed.

Fibronectin has been reported to utilize an RGD

(ArginineGlycineAspartic acid) binding motif for its

binding to integrins (specifically 51) localized at theplasma membrane of macrophages to induce IL1

production. To assess the role of exosomeassociated

fibronectin on IL1 induction, commercially available

RGD sequence mimics were used. Incubation of OEX

withTHP1cellsresultedintheproductionandreleaseof

IL1. The negative controlmimic peptide (CP),which

does not block integrin binding of fibronectin, was

included as a negative control. In Figure 5, CP was

observednottoexhibitanyeffectonIL1 productionor

release. In contrast, the twomimic peptides (ANT and

BFCV),knowntoinhibitfibronectinbindingtointegrins,

significantly suppressed IL1 release by THP1 cells(p


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Figure5.Exosomeassociatedfibronectinmediatestheincreasedreleaseof IL1.HumanTHP1werecultured inculturemedia

only (THP) or precultured with the various RGD mimic

peptidesat0.5mg/mlfor30minutes.OEX(exosomesfromULO

cells) were added at 100g/ml for 6 hours, followed by 30

minuteswith orwithoutATP (1mM) at 37C. Supernatants of

cultureswereremovedtodetermine thequalityofIL1 protein

releasedbyELISA.Means and standarddeviations from threeindependent studies are shownwithpvalues calculatedusing

Studentsttest.***indicateslevelofsignificance(p


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fromvesiclesobtained fromovariancancerpatientswas

demonstrated by mass spectrometric sequencing and

confirmedbyWesternimmunoblotting(Figures2and3).

The ability of this exosomeassociated fibronectin to

inducethereleaseofIL1 wasfurtherinvestigated.Our

studies demonstrate the exposure of THP1 cells totumourderived exosomes induced the significant

production and release of proinflammatory

cytokines/chemokines, includingofparticularnote IL1

(Figure4).

In vitro, fibronectin stimulates the expression of IL1mRNA and its translation into the 31kDa intracellular

precursor protein, along with secretion of the 17kDa

activeform inhumanmononuclearcells [31].Thiseffect

offibronectinismediatedbythespecificcellsurface51

integrin receptor, which activates poorly understood

intracellular signals to induce IL1 expression [32].Fibronectin contains a sequence, termed ArgGlyAsp

(RGD), which promotes its attachment to integrin

receptors.Monocytesandmacrophageshavebeenshown

topossess fibronectin receptors that recognize theRGD

motif and mediate proinflammatory cytokine

production.The effectof fibronectinhasbeen shown to

be dependent on binding of the RGD sequence of

fibronectin to integrin receptors, as this effect couldbe

inhibited by integrin receptor blocking peptides (anti

RGDsequencemimics)[33].

IL1 posttranslationalprocessingandreleaseisahighlyregulated pathway utilizing a multiprotein complex,

termedthecapase1inflammasome[34].IL1 isinitially

synthesizedasa34kDprecursorproteinthatiscleavedto

the biologically active 17kD form by active caspase1.

Inactive procaspase1 is constitutively present in both

M1 and M2 macrophages and is activated within M1

macrophages by selfcleavage, occurring in the

inflammasomecomplex.Thenucleotidebindingdomain

andleucinerichrepeatreceptorcontainingpyrindomain

3 (NLRP3) inflammasome can be activated by specific

microbialmotifs,bymicrobialtoxins,byuricacidcrystals

andby extracellularATP acting through theATPgated

plasma membrane ion channel, the P2X7 receptor.

Addition of extracellular ATP to macrophages can

generallyincreasethereleaseofmatureIL1.Incontrast

to our previous study using trophoblastderived

exosomes, tumourderived exosomes do not require

additionofATPtoinducereleaseofIL1(Figure5).Itis

unclearwhether these exosomes carryATP or utilize a

distinctpathwaytoinducetheproductionandreleaseof

IL1. However, these possibilities are currently under

investigation.

The IL1 promoter region contains AP1 and cJun

transcription factor binding sites that are critical tomediate certain pathologic conditions. Our results

demonstrate that tumour exosomes induce IL1 and

produce phosphorylation ofAkt2 and cJun (Figure 6).

The addition of the RGD mimics resulted in the

suppression of Akt2 and cJun phosphorylation,

following addition of ULO exosomes. These findings

confirm the essential role of exosomeassociatedfibronectin in the regulatory events leading to the

promotionofIL1 productionandrelease.

While other studies have investigated the role of

fibronectin and other components of theECMs on the

inductionofIL1,inthecurrentstudy,wedemonstrate

IL1 inductionby fibronectinassociatedwith tumour

derived exosomes. This study with ovarian cancer

derived exosomes demonstrates that these tumour

exosomes also express fibronectin,whichmay account

fortheproinflammatorymicroenvironmentobservedin

cancer. Exposure of macrophages to ovarian tumourderived exosomes appears to educate these cells to

M1macrophages.TheseM1macrophagesproducepro

inflammatory cytokines and chemokines, including

TNF,IL8,MCP1(CCL2)andIL1.Whileenhanced

levelsof IL1characterizeM1polarizedmacrophages,

IL1 is absence in M2 polarized macrophages [24].

These findingsmayhave implications for thepotential

use of exosomeassociated fibronectin as potential

biomarkers for cancers.Exosomeassociated fibronectin

may also serve as a therapeutic target for cancer,by

targeting the fibronectininduced proinflammatory

environment essential for tumour growth andproliferation.

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Ovarian Cancer Derived Exosomal Fibronectin Induces Pro Inflammatory Il 1beta