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: ddtaylor@louisville.edu
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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ARTICLE
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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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