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MITOTICARRESTAFFECTSCLUSTERINGOFTUMORCELLS2

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4 Julia BONNET1, Lise RIGAL1, Odile MONDESERT1, Renaud MORIN2, Gaëlle5

CORSAUT1,MathieuVIGNEAU1,BernardDUCOMMUN1,3andValérieLOBJOIS1*6

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1UniversitédeToulouse,ITAV,CNRS,Toulouse,France8

2IMACTIV-3DSAS,Toulouse,France9

3CHUdeToulouse,Toulouse,France10

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*Correspondingauthor:15 16 ValérieLOBJOIS17

ITAV-USR3505,1placePierrePotier,18

F-31106ToulouseCedex1,France19

Tel:+3358299104720

E-mail:valerie.lobjois@itav.fr 21

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ABSTRACT1

Background2

Cancer cell aggregation is akeyprocess involved in the formationof tumorcells3

clusters.Ithasrecentlybeenshownthatclustersofcirculatingtumorcells(CTCs)4

haveanincreasedmetastaticpotentialcomparedtoisolatedcirculatingtumorcells.5

Several widely used chemotherapeutic agents that target the cytoskeleton6

microtubulesandcausecellcyclearrestatmitosishavebeenreportedtomodulate7

thenumberofCTCsorthesizeofCTCclusters.8

Results9

Inthisstudy,weinvestigatedinvitrotheimpactofmitoticarrestontheabilityof10

breast tumorcells to formclusters.Byusing live imagingandquantitative image11

analysis, we found that MCF-7 cancer cell aggregation is compromised upon12

incubationwithpaclitaxelorvinorelbine,twochemotherapeuticdrugsthattarget13

microtubules. In linewith these results,we found thatMCF-7breast cancer cells14

experimentally synchronized and blocked in metaphase aggregated poorly with15

stronglyreducedcohesion.Tomonitorclusteringat thesingle-cellscale,wenext16

developed and validated an in vitro assay based on live video-microscopy and17

custom-designedmicro-devices.The studyofcluster formation fromMCF-7cells18

thatexpressthefluorescentmarkerLifeAct-mCherryusingthisnewassayallowed19

showing that substrate anchorage-independent clustering of MCF-7 cells was20

associatedwiththeformationofactin-dependenthighlydynamiccellprotrusions.21

Metaphase-synchronizedandblockedcellsdidnotdisplay suchprotrusions, and22

formedverylooseclustersthatfailedtocompact.23

Conclusions24

3

Altogether,ourresultssuggestthatmitoticarrestinducedbymicrotubule-targeting1

anticancer drugs prevents cancer cell clustering and therefore, could reduce the2

metastaticpotentialofcirculatingtumorcells.3

4 5

Keywords: Cancer cell clustering, mitosis, microtubule-targeting agents,6 anchorage-independentaggregation,quantitativeliveimaging.7 8

9

4

BACKGROUND1 2 Metastaticdisseminationofepithelialtumorcellsthatinvade,circulate,andforma3

tumoratdistantsites[1,2]isamajorchallengeforcancertherapy.Circulatingtumor4

cells (CTCs)aredetected inpatients’blood samples, andCTCclustershavebeen5

associatedwithhighermetastaticpotential [3,4]. Indeed, formationof tumorcell6

clusterspreventsanoikis in theabsenceof anchorageandprolong their survival7

[5,6].Moreover,CTCclustersdisplayhighermetastaticpotentialthanisolatedCTCs8

andareassociatedwithadverseoutcomes[3,7].Theirroleintumordissemination9

suggest that they should be considered in anti-metastasis strategies [8-10].10

Therefore, the clinical implementation of sensitive and reliable technologies to11

detectandquantifyCTCsandCTCclustersiscurrentlythesubjectofmajorinterest12

(see for instance [11-13]).However,only fewregulatorsof tumor cell clustering13

havebeenidentified,andtheunderlyingmechanismsremainunclear.Forinstance,14

plakoglobin,acelljunctioncomponent,isdifferentiallyexpressedinbreastcancer,15

its knockdown in mouse abrogates CTC cluster formation, and is a significant16

prognostic predictor [3,14]. Similarly, it has been shown that breast cancer17

metastasesarise fromkeratin14-expressing tumorcell clusters [1].We recently18

reported,usingtime-lapsemicroscopy-basedclusteringthatE-cadherinsandalso19

desmogleinanddesmocolin,twodesmosomalproteins,areinvolvedincancercell20

aggregation[15].Usingthesameapproach,wealsodemonstratedtheinvolvement21

ofgapjunctionintercellularcommunicationinregulatingtheearlieststepoftumor22

cellclustering[16].23

Cellproliferationistightlyassociatedwiththesuccessfulcompletionofthecellcycle24

that culminates with mitosis. Anti-mitotic drugs that impair or inhibit mitosis25

5

ultimatelyresultincelldeath,andeffectivelytargetandkilltumorcells [17].Spindle1

poisons,suchasvincaalkaloids,paclitaxelandrelatedtaxanes,targetmicrotubule2

dynamics,resultinginmitoticarrestthroughtheactivationofthespindleassembly3

checkpoint [18].These compoundsarehighlyeffective anti-cancerdrugs in vitro4

andinclinicalsettings,andarecurrentlyusedtotreatmanytumortypes,including5

breastandovarianmetastaticcancer[19-21].Interestingly,veryrecently,ascreen6

of a FDA-approved compounds library identified tubulin polymerizing inhibitors7

fortheirabilitytodecreasethesizeofhumanbreastCTCclusters[9].However,a8

pro-metastatic effect of these compounds has also been reported in mouse9

mammary tumor models associated with an increase of circulating tumor cells10

[22,23]. Indeed, thenumberofdetected isolatedcirculatingtumorcells increases11

massivelyafterpaclitaxeltherapyindicatingthattumorcellsaremobilizedfromthe12

primarytumorinresponsetochemotherapy.13

Considering these data, we wondered whether tubulin polymerizing inhibitors,14

throughtheiranti-mitoticeffect,couldimpacttheabilityoftumorcellstoaggregate15

andformclusters.16

WefirstobservedthatpaclitaxelandvinorelbineaffectinvitroformationofMCF-717

clusters. We then used population and single-cell scale in vitro assays with18

experimentally blockedMCF-7 cells atmetaphase and found thatmitoticMCF-719

breastcancercellsformclustersthatarepoorlyaggregatedandunstructured.This20

impairedclusteringwasassociatedwithroundingofmitoticcellsandlackofactin-21

basedmembranedynamics.Wethereforeproposethatanticancerdrug-inducedcell22

cycle blockade atmitosismightmodulate themetastatic potential of circulating23

tumorscellsbyreducingtheirclustering. 24

6

RESULTS 1 2 MCF-7cellstreatedwithpaclitaxelorvinorelbineless efficiently form clusters3

Paclitaxel and vinorelbine are two microtubule targeting anticancer drugs that4

inducecellcycleblockatmitosisandinhibitionofcellproliferation[20].Toassess5

theireffectonMCF-7cellability tocompactandformclusters,cells treatedwith6

paclitaxel or vinorelbine for 24 hourswere subjected to a previously described7

aggregationassayinwhichtheprogressiveaggregationandcompactionof500cells8

seeded innon-adherent96-wellplatesaremonitoredbyvideo-microscopy for59

hours [15,16]. Cells treatedwith 100 nMpaclitaxel or 20 nM vinorelbine for 2410

hours accumulated inmitosiswith very limited cell death, as confirmed by flow11

cytometryanalysis(datanotshown).Figure1ashowsrepresentativeimagesofthe12

clustering and compaction kinetics of control and treated cells. To quantify13

compaction over time, cluster's area determination at each time point was14

performedbyautomated imagesegmentation(red line)usingacustom-designed15

MATLABroutine.Usingthisquantification,wefoundthatclusteringofMCF-7cells16

treatedwithpaclitaxelorvinorelbinewasaltered,andthatat5hours,compaction17

wasreducedbyabout25%comparedwithuntreatedcells(Fig.1b,c).18

19

Clusterformationisreducedinmetaphase-synchronizedMCF-7breastcancer20

cells.21

Considering the observed reduction of cancer cells compaction induced by22

paclitaxelandvinorelbine,weexaminedtheeffectofmitoticarrestonthecancer23

cellabilitytoformclusters.WefirstmonitoredclusterformationinMCF-7breast24

cancercellssynchronizedandblockedinmetaphase.Tothisaim,weincubatedcells25

7

withnocodazole for20hours followedby culturewith theproteasome inhibitor1

MG132for2hourstosynchronizecellsatthemetaphase-anaphasecheckpointand2

byafinalshake-offsteptoretainonlymitoticcells[24](seeMethodsandAdditional3

file 1: Fig. S1 for details). Flow cytometry analysis of the cell cycle distribution4

performedafter theshake-off indicated that about85%of cellswerearrested in5

metaphase.Thiswasalsoconfirmedbyimmunofluorescenceanalysiswithananti-6

a-tubulinantibodythatshowedmetaphasearrestedcellswithanorganizedbipolar7

mitoticspindle(Additionalfile1:Fig.S1b,c).8

Then, we used the same aggregation assay. Figure 2a shows representative9

micrographs of the aggregation kinetics of control (no synchronization) and10

metaphase-synchronizedMCF-7 cells. The quantification (Fig. 2b) of these data11

indicatedthatMCF-7cellclusteringwasmuchslowerandcellcompactiontoform12

clusterwas reduced by approximately 50% inmetaphase-blockedMCF-7 tumor13

cellscomparedwithcontrolcells.14

15

Cohesionofmetaphase-blockedMCF-7cellaggregatesisstronglyreduced16

As observed in Figure 2, aggregates formed by metaphase-blocked cells after 517

hourswerelessroundthanthoseformedbycontrolasynchronouscells.Thiswas18

confirmedbythe findingthat theaggregatecircularity(Fig.3a)wassignificantly19

lowerinmetaphase-blockedthanincontrolMCF-7tumorcells(meanvalue:0.38±20

0.1versus0.87±0.02).Thissuggeststhatmetaphase-blockedcellsaggregatemore21

slowlythancontrolcellsandformlooserandlesscohesiveclusters.Therefore,we22

assessed the aggregate cohesion by using a dissociation assay in which the cell23

aggregatecohesionismechanicallychallengedbyaspecificnumberofsequential24

8

aspirationsand flushesperformedusingamicropipette.Weused thenumberof1

releasedcellsasanindicatoroftheaggregatecohesion(seeMethodsfordetails).2

The number of cells released from control cell aggregates was small (Fig. 3b),3

indicating that strong intercellular interactionswere already established after 54

hours. Conversely, metaphase-blocked cell aggregates were rapidly and fully5

dissociated, indicating that the less efficient aggregation of metaphase-6

synchronizedcellswasassociatedwiththe formationof lesscohesiveclustersby7

poorlyadherentcells.8

9

Anoriginalassaytoinvestigateclusteringatthesingle-cellscale.10

Thepreviousaggregationassaydoesnotallowassessingcell-cellinteractionatthe11

single-cellscaleduetothehighcelldensity.Therefore,wedevelopedanewvideo-12

microscopyassaytoanalyzethedynamicsofanchorage-independentclusteringin13

singleMCF-7cells.Tothisaim,wedesignedandproduceddedicatedPDMSmicro-14

wells(seeMethodsandAdditionalfile2:Fig.S2)thatweretreatedwithpluronic15

acid, a non-ionic surfactant toprevent cell anchorage to the substrate. Then,we16

seededMCF-7cellsthatexpresstheLifeAct-mCherryfluorescentreporterinthese17

micro-wells at a concentration that allowed the sedimentation of about 15-2018

cells/microwell (Fig. 4a). Time-lapse acquisition (see Additional file 3:19

SupplementaryMovie1andrepresentativefluorescenceimagesinFig.4a)showed20

thatwithin3hours,untreatedcellsformedseveralsmallclustersthatprogressively21

gatheredtogether,resultinginamainsinglecompactcluster.Intheseexperimental22

conditions,weobservedthatduringaggregation,controlMCF-7cellsformedlarge23

and highly dynamic protrusions that could be visualized by video-microscopy24

9

thanks to the LifeAct-mCherry fluorescent reporter (Fig. 4b, left panels and1

Additionalfile4:SupplementaryMovie2).Thedynamicsoftheselargeprotrusions2

could be captured by determining the aspect ratio of single cells that were3

individually analyzed during the first hour of the clustering experiment (Fig. 4b,4

rightpanel).Fromthesedata,wedeterminedthemeanvalue(µ)oftheaspectratio5

(AR)andthestandarddeviation(s)of theARvaluesofeachcellduringthe first6

hourasanindicatorofthecellshapechangesovertime.7

8

Protrusions associated with anchorage-independent self-aggregation are9

dependentontheactincytoskeleton.10

Consideringthecrucialroleofactinfilamentpolymerizationindrivingcellshape11

dynamicsandmotility,weincubatedMCF-7cellswithlatrunculinA,whichdisrupts12

microfilamentorganizationbybindingtomonomericG-actin,andwithCK666,an13

inhibitoroftheF-actinnucleatorcomplexArp2/3[25].Inasynchronous(control)14

cells, latrunculin A and CK666 abolished cell protrusion formation (Fig. 5a, and15

Additonalfile5:SupplementaryMovie3andAdditonalfile6:SupplementaryMovie16

4)andprotrusiondynamics,asindicatedbythesignificativedecreaseofthemean17

andstandarddeviationoftheARcomparedwithuntreatedcells(Fig.5b).Moreover,18

attheendofthe3-hourclusteringexperiments,cellaggregationwasimpairedin19

asynchronous cells incubated with CK666 or latrunculin A, as shown by the20

significantlyhigherARandlowercircularityoftheformedclusters(Fig.5c).These21

results indicate that inhibition of actin-dependent cell protrusion formation and22

dynamicsisassociatedwithlessefficientclustering.23

10

Metaphase-blocked MCF-7 cells do not develop large protrusions during1

anchorage-independentaggregation.2

In themicrodevice-based assay,metaphase-synchronized cells started to form a3

cluster, but they did not organize and efficiently compact (Fig. 6a). Quantitative4

analysisoftheshapeofthefinalclusterineachmicrowell(Fig.6b-c)bydetermining5

itsaspectratioandcircularityshowedthatcontrol(asynchronized)cellsformeda6

compact structure (mean aspect ratio of 1.6 and mean circularity of 0.5 for7

untreatedcells)aswellascellsincubatedonlywithnocodazoleorMG132(Fig.6c8

andAdditionalfile7:Fig.S3aandS3b).Conversely,metaphase-blockedcellsformed9

unstructuredclusters(meanaspectratioof2.5andmeancircularityof0.3).Inthese10

conditions, we observed that during anchorage-independent aggregation11

metaphase-blocked cells did not form dynamicmembrane protrusions (Fig. 6d).12

Comparison of the AR in control (asynchronous) andmetaphase-arrested single13

cells(Fig.6e)confirmedthisobservation. IncubationwithnocodazoleorMG-13214

alonedidnotaffectprotrusionformation(Additonalfile7:Fig.S3candS3d).15

These results clearly show that, in these experimental conditions, metaphase-16

blockedcellsaggregatesignificantlylessefficientlyandformlooseclustersofpoorly17

associated cells. As shownwith actin-targeting drugs, less efficient clustering is18

associatedwithabsenceofdynamiccellprotrusionsformation.19

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DISCUSSION1 2

Duringtheprocessofmetastasis,cancercellsescapefromtheprimarytumorand3

reachdistantsitesthroughthebloodorlymphaticvessels.Thosecellsareknownas4

circulatingtumorcells(CTCs).Furthermore,clustersofcirculatingtumorcellshave5

alsobeendetectedinthebloodofpatientswithmetastaticcancers.Theseclusters6

haveanincreasedmetastaticpotentialcomparedtosingleCTCsandtheirpresence7

correlateswithpoorprognosis[3].Arecentstudyidentifiedareductioninsizeof8

CTC clusters after treatmentwith tubulin binding drugs [9]. Several studies also9

revealedvariationof thenumberofCTCsafter treatmentswithanticancerdrugs10

that target themicrotubule cytoskeleton [22].Altogether thesedata suggest that11

treatments with these anti-mitotic drugs might have an impact of CTC clusters12

formationandstability.13

In this study, we investigated in vitro how anti-mitotic agents could impact the14

clusteringandaggregationofmitoticMCF-7cancercellsinanchorage-independent15

conditionsusingdedicatedmicrodevicesandtime-lapsevideomicroscopy.16

First,byvideo-microscopyanalysisoftheaggregationandcompactionofalargecell17

population over time, we showed that anchorage-independent aggregation is18

inhibitedbypaclitaxelandvinorelbinetreatmentsaswellasinmetaphase-blocked19

mitotictumorcells.Then,usingamechanicalassay,wefoundthatcohesionofsuch20

cellaggregatesisdramaticallyreduced.Finally,wedevelopedanewassayinwhich21

an array of PDMSmicro-wells allowsmonitoring and studying the clustering of22

singlecancercellsintheabsenceofanchorage.Intheseconditions,again,wefound23

thatMCF-7cellaggregationislessefficientanddonotresultincompactaggregates,24

12

but rather in loose clusters. These results strongly suggest that in MCF-7 cells1

blockedinmitosis,thecapacitytoaggregateandformcompactclustersisreduced.2

Weshowedthatduringanchorage-dependentclustering,MCF-7cellsformlargeand3

dynamic actin-dependent protrusions, and that alteration of their formation by4

usingactin-targetingdrugsaltersclusterformationinPDMSmicro-wells.Thisresult5

is consistent with our previous findings showing that latrunculin A affects cell6

aggregationinultra-lowattachmentplates[16].Giventhefactthatourexperiments7

wereperformedusingultralowattachmentplatesonwhichcellscouldnotadhere,8

themodificationsofcellshapeduringtheclustersformationcouldnotbeattributed9

to adherence to the substratum and migration. MCF-7 cells synchronized and10

blocked inmetaphasedonot formprotrusions.Roundingatmitosis isassociated11

withelevated intracellularpressureand recruitmentofmyosin to the cell cortex12

thatleadstoanincreaseincorticaltension,whichismaximalatmetaphase[26,27].13

Moreover,wepreviouslydemonstratedthatcytoskeletontensionduetomyosinIIa14

actsasan inhibitorofcellaggregation[15].Therefore,wehypothesizedthat less15

efficientclusteringofmetaphase-synchronizedcellscouldbeduetoadecreasein16

formation and/or dynamics of protrusions, as a consequence of cortical tension17

increase.Accordingly, theabsenceofprotrusion inMCF-7cellssynchronizedand18

blockedinmetaphasesuggeststhatacto-myosindependentcorticaltensioncould19

alsobeanimportantparameterintumorcellclusterformation.20

Dynamic microtubule-based protrusions, named micro-tentacles, have been21

describedalsoinbreastcarcinomacelllines[28].Theformationoftheseextensions22

iscounteractedbytheactin-cytoskeletonandthesemicrotubule-dependentplasma23

membraneextensionshavebeenassociatedwithtumorcellretention inthe lung24

13

vasculature. Here, we found that nocodazole does not affect actin-dependent1

protrusion formation and aggregation dynamics in MCF-7 cells, showing that2

different mechanisms involving the plasma membrane could contribute to the3

survival andmetastatic potential of CTCs during their journey from the primary4

tumortothevasculature,clustering,andformationofsecondarymetastases.5

TheprognosticvalueandclinicalrelevanceofCTCenumerationanditsassociation6

withprogression-freesurvival(PFS) inadvanced-stagebreastcancer isnowwell7

establishedandthepresenceofCTCclustershasbeenassociatedwithshorterPFS8

[29].CTCclustersprovideadditionalprognosticvalue comparedwithCTCcount9

alone[30-32].Theeffectsofmicrotubule-targetedagentsonCTCbiologyisnotfully10

understood. As stated above, several publications showed that CTC number11

increasesmassivelyupon therapy, suggestingthat they canbe rapidlymobilized12

fromtheprimarytumoraftertreatmentwithpaclitaxel[22].Asdiscussedbyothers,13

this should be carefully examined to ensure that antimitotic therapies do not14

increasethemetastaticpotential[28].Ithasbeenreportedthattaxanetreatment15

enhances tumor cell reattachment [33]. However, to date no data has been16

published on the increase of isolated CTC and clusters upon treatment with17

paclitaxel and related compounds [22]. On the basis of our findings, we might18

hypothesize that paclitaxel therapy while leading to CTC rapid increase could19

decreasetheircapacitytoformclusters,thusresultinginanoverallreductionofthe20

metastaticrisk.21

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CONCLUSIONS1 2

In summary, metaphase-blocked cells initiate spontaneous aggregation in3

anchorage-independentinvitroexperimentalconditionsbutfailtoformcompact4

clusters and associated dynamic actin-dependent protrusions. In linewith these5

results, treatments with the anticancer tubulin-targeting drugs paclitaxel and6

vinorelbine alter MCF-7 tumor cell clusters formation. These results provide7

insights on the potential effect of anti-mitotic chemotherapeutic agents on the8

metastaticpotentialofCTCs.9

10 METHODS11 12

Cellculture13

MCF-7 cells (ATCC HTB-22) were cultured in RPMI (Gibco, Life Technologies)14

supplementedwith 1µmol/L insulin (SigmaAldrich), 10% fetal calf serum (FCS)15

(Gibco,LifeTechnologies)and1%penicillin/streptomycin(100U/ml,Gibco,Life16

Technologies) in a humidified atmosphere of 5% C02 at 37 °C. For time-lapse17

microscopyimaging,culturemediumwasreplacedbyOPTIMEM+Glutamax(Gibco18

byLifeTechnologies)supplementedwith1µmol/L insulin,10nmol/Lß-estradiol,19

20ng/ml epidermal growth factor (Invitrogen), B-27 Supplement (1X,20

Invitrogen),1%penicillin/streptomycin(100U/ml,Gibco,Lifetechnologies).21

Tostudythe impactofpaclitaxelandvinorelbine,cellswere incubated inculture22

medium containing 100 nM paclitaxel or 20 nM vinorelbine for 24 hours, then23

trypsinized and seeded for aggregation assay in culture medium containing the24

sameconcentrationsofpaclitaxelandvinorelbine.Forsynchronizationinmitosis,25

15

cells were incubated with 200 ng/ml nocodazole for 20h to accumulate in an1

abnormal pro-metaphase state. Cells were then incubated in culture medium2

containing200ng/mlnocodazoleand25µMMG132for30min,andtheninmedium3

containingonly25µMMG132for1.5hours,accordingtotheprotocoldescribedby4

Cazalesetal.[24].AdditionofMG132blockedcellsinmetaphasebyinhibitingsister5

chromatidseparation.Mitoticshake-off [34]wasusedtoselectonlymitoticcells6

thatwereusedfortheclusteringandaggregationassays.Forcontrolexperiments,7

MG132(25µM)ornocodazole(200ng/ml)wereaddedtotheculturemediumjust8

beforeseedingforaggregationassays.Foractincytoskeletoninhibition,latrunculin9

A(200nM,Sigma)andCK666(150µM,Sigma)wereaddedtotheculturemedium10

justbeforeseedingforaggregationassays.11

Immunofluorescentstaining12

Cellsweregrownoncoverslipscoatedwithpoly-L-lysine.CellswerewashedinPBS,13

fixedfor10mininformalin(Sigma)thenwashedandpermeabilizedinPSB/0.25%14

TritonX-100for5minatroomtemperature,andincubatedinPBS/1%BSA30min15

at room temperature. Coverslipswere then incubated 1 hour at 37°Cwith anti-16

tubulinantibodies(1:2000,Sigma)inPBS/0.1%BSA.AfterwashesinPBS,goatanti-17

mouseAlexa488antobodies(1/800,Molecularprobes)wereappliedfor1hourat18

room temperature. DNA was stained with DAPI at 1µg/ml for 10 min at room19

temperature.20

LifeAct-mCherry-expressingMCF-7cellline21

The 17 amino acid LifeAct coding sequence fused toGFP2was excised from the22

pLifeAct-TagGFP2vector(Ibidi;catalognumber#60101)andclonedinthepTRIP23

lentiviralshuttlevectorinframewiththecDNAencodingthemCherryfluorescent24

16

protein. The resulting plasmid (pTRIP LifeAct mCherry) was used to produce1

lentiviral particles in 293FT embryonic kidney cells (LifeTechnologies) after2

calciumchloridetransfectionwiththepGag/polandpVSV-Gplasmids(providedby3

theVectorologyplatform,INSERMU1037)[35].At7hourspost-transfection,DMEM4

+Glutamax(GibcobyLifeTechnologies)complementedwith10%FCSwaswashed5

out and replaced with serum-free OPTIMEM + Glutamax (Gibco by Life6

Technologies).Lentiviralparticleswereharvested48hourslaterandtheviraltiter7

was quantified by flow cytometry (BD Accuri C6) in HT1080 cells (ATCC)8

transducedwithserialdilutionsoflentiviruses.MCF-7cells(ATCCHTB-22)were9

then transduced in the presence of 4μg/ml protamine sulfate in OPTIMEM +10

Glutamax. The medium was replaced 7 hours later by RPMI (Gibco by Life11

Technologies)supplementedwith10%FCSand1µM insulin (Sigma-Aldrich,Ref.12

I0516). The generated stable LifeAct-mCherry-expressing MCF-7 cell line13

underwenttworoundsofcellsorting(CytometryandCellSortingplatform,INSERM14

UMR1048)followedbysingle-cellclonalisolationin96-wellplates.15

Flowcytometryanalyses16

Trypsinizedcellswerecollectedandfixedin4%formalinsolution(Sigma-Aldrich)17

for10min,thenwashedandpermeabilizedinPBS/1%BSAcontaining0.25%Triton18

X-100onicefor5min.Mitoticcellsweredetectedwiththe3.12.i.22antibody[36]19

diluted(1:10000)inPBS/0.1%BSA.AfterawashinPBS,cellswereincubatedwith20

a goat anti-mouse Alexa Fluor 488 antibody (Molecular Probes) at room21

temperature for1h.AfterDNAstainingwith10μg/mLpropidium iodide (Sigma22

Aldrich)atroomtemperaturefor20min,cellswereanalyzedwithanAccuri™C623

FlowCytometer(BDScience)andtheAccurisoftware.24

17

Aggregationassay1

Thisassaywasperformedessentiallyaspreviouslydescribed [15,16].Cells (5002

cells/well) were seeded in low-attachment round-bottomed 96-well plates3

(Costar®), except in the 36 peripheral wells to avoid edge effects. Plates were4

centrifugedat400gfor4min,andthencellaggregationineachwellwasfollowed5

bytime-lapsevideo-microscopy.Imageswereacquiredwithaninvertedwidefield6

ZeissAxioObservermicroscopefittedwitha0.3N.A.10XobjectiveandaCoolSNAP7

CDDcamera(Roperscientific)inbright-fieldforatleast5h(1acquisition/15min).8

Ateachtimepointandposition,20-µmspacedz-stacksover160µmdepth(8stacks)9

in brightfield were acquired. A custom-made MATLAB procedure was used to10

monitor and measure cell cluster formation over time. The main steps of the11

workflowwere: (1) imageprocessingat each timepointand foreach clusterby12

focusstackingtomergeimagesofmultiplefocalplanesintoonein-focusimage;(2)13

binarizationandedgedetectionwithaSobelfiltertodefinetheboundariesofeach14

clusterandofholesinsidethecluster(toexcludethem);(3)savingtheprojection,15

segmentation and image overlay; and (4) calculation of the typical parameters16

(perimeter,area,normalizedarea:AreaT0/AreaT(x)).17

Evaluationofaggregatecohesion-Flushassay18

Aggregatesformedineachwellweremechanicallydissociateddirectlyinthewells19

bygentlyaspirating50µlofcellsandmediumwithamulti-channelmicropipetteand20

vigorouslyflushingback(1,3,5or8flushes).Cellswerethenallowedtosediment21

for10minbeforequantifyingthenumberofdissociated individualcells in10µL.22

Quantificationwasdoneintriplicateforeachexperimentalcondition.23

Single-cellclusteringassayindedicatedPDMSmicro-wells24

18

ThePDMSpre-polymerwasmixedwiththepolymerizationagentSylgard184(10:11

ratio), degassed in a vacuum chamber, and poured in a siliconwafer (RENATER2

facilityofLAAS,CNRS,France).Afteraseconddegassing,PDMSwascuredat60°C3

overnight.ArraysofninePDMSmicro-wells(seeAdditionalfile2:Fig.S2)werecut,4

peeled off, and glued in each compartment of CELLviewTM cell culture dishes5

(Greiner Bio-one). Micro-wells were incubated with 20 mg/ml Pluronic-F1276

(Sigma)overnighttopreventcelladhesion,andthenrinsedtwicebeforeuse.7

LifeAct-mCherry-expressingMCF-7cellsweredistributedinthecompartmentsata8

density thatallowedthesedimentationofapproximately20cellspermicro-well.9

Clusterformationwasfollowedbytime-lapsevideomicroscopyusinganinverted10

widefield Zeiss Axio Observer microscope fitted with a 0.3 N.A. 10X objective.11

Imageswereacquiredfor3h(oneacquisitionevery10s)andprocessedwithlmageJ12

software packages [37]. Before automated analysis, images were manually13

corrected. Specifically, partsof other cells, staining background and debriswere14

removedusingtheclearingfunctionofImageJ.Then,thelmageJmacrowasusedfor15

imagesegmentationandcalculationoftheshapedescriptors(circularityandaspect16

ratio).17

Statisticalanalysis18

Datawere analyzedwithGraphPadPrismversion6.00 (GraphPadsoft-ware,La19

JollaCaliforniaUSA,www.graphpad.com)20

21 ABBREVIATIONS 22 23 CTC:Circulatingtumorcell24 PDMS:Polydimethylsiloxane25 26 27

19

DECLARATIONS1 2

Ethicsapprovalandconsenttoparticipate3

Notapplicable4

Consentforpublication5

Notapplicable.6

Availabilityofdataandmaterials7 8 All data generated or analysed during this study are included in this published9

articleanditssupplementaryinformationfiles.10

CompetingInterests11

Theauthorsdeclaretheyhavenocompetinginterests.12

Funding13

ThisworkwasgenerouslysupportedbyagrantfromlaLigueNationaleContrele14

Cancer(ComitédelaHaute-Garonne).15

Author'scontributions16

JB, BD and VL conceived the experiments. JB, LR, OM and GC conducted the17

experiments.MVandRMdevelopedtheimageprocessingalgoithms.JB,LR,BDand18

VL performed analysis and interpretation of the data. JB, BD and VL wrote the19

manuscript.Allauthorsreviewedthemanuscript.20

Acknowledgements21

Wearegratefultothemembersofourgroupfortheirinterestinthisproject.The22

supportbyITAVandtheTRI-Genotoulimagingfacilityisgratefullyacknowledged.23

JBissupportedbyaPhDfellowshipfromtheRégionOccitanie.Theauthorsthank24

ElisabettaAndermarcherforexpertmanuscriptediting.Thisworkwasgenerously25

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supportedbyagrantfromlaLigueNationaleContreleCancer(ComitédelaHaute-1

Garonne).2

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16. GavaF,RigalL,MondesertO,PesceE,DucommunB,LobjoisV.Gapjunctions1 contributetoanchorage-independentclusteringofbreastcancercells.BMCCancer2 2018;18(1):221.3 17. Rao CV, Kurkjian CD, Yamada HY. Mitosis-targeting natural products for4 cancerpreventionandtherapy.CurrDrugTargets2012;13(14):1820-30.5 18. Matson DR, Stukenberg PT. Spindle poisons and cell fate: a tale of two6 pathways.MolInterv2011;11(2):141-50.7 19. Gourmelon C, BourienH, AugereauP, Patsouris A, Frenel JS, CamponeM.8 Vinflunine for the treatment of breast cancer. Expert Opin Pharmacother9 2016;17(13):1817-23.10 20. JordanMA,WilsonL.Microtubulesasatargetforanticancerdrugs.NatRev11 Cancer2004;4(4):253-65.12 21. LebertJM,LesterR,PowellE,SealM,McCarthyJ.Advancesinthesystemic13 treatmentoftriple-negativebreastcancer.CurrOncol2018;25(Suppl1):S142-S50.14 22. AdachiY,YoshimuraM,NishidaK,UsukiH,ShibataK,HattoriM,etal.Acute15 phase dynamics of circulating tumor cells after paclitaxel and doxorubicin16 chemotherapy in breast cancer mouse models. Breast Cancer Res Treat17 2018;167(2):439-50.18 23. ChangYS,JalgaonkarSP,MiddletonJD,HaiT.Stress-induciblegeneAtf3in19 thenoncancerhost cells contributes to chemotherapy-exacerbatedbreast cancer20 metastasis.ProcNatlAcadSciUSA2017;114(34):E7159-E68.21 24. CazalesM, BoutrosR, BrezakMC, ChaumeronS, PrevostG,DucommunB.22 Pharmacologic inhibitionofCDC25phosphatases impairs interphasemicrotubule23 dynamicsandmitoticspindleassembly.MolCancerTher2007;6(1):318-25.24 25. FritzscheM,LiD,Colin-YorkH,ChangVT,MoeendarbaryE,FelceJH,etal.25 Self-organizingactinpatternsshapemembranearchitecturebutnotcellmechanics.26 NatureCommunications2017;8(1):14347.27 26. Ramanathan SP, Helenius J, Stewart MP, Cattin CJ, Hyman AA, Muller DJ.28 Cdk1-dependentmitoticenrichmentofcorticalmyosinIIpromotescellrounding29 againstconfinement.NatCellBiol2015;17(2):148-59.30 27. StewartMP,Helenius J, ToyodaY, Ramanathan SP,MullerDJ, HymanAA.31 Hydrostaticpressureandtheactomyosincortexdrivemitoticcellrounding.Nature32 2011;469(7329):226-30.33 28. MatroneMA,WhippleRA,ThompsonK,ChoEH,VitoloMI,BalzerEM,etal.34 Metastatic breast tumors express increased tau, which promotes microtentacle35 formation and the reattachment of detached breast tumor cells. Oncogene36 2010;29(22):3217-27.37 29. Paoletti C, Schiavon G, Dolce EM, Darga EP, Carr TH, Geradts J, et al.38 CirculatingBiomarkersandResistancetoEndocrineTherapyinMetastaticBreast39 Cancers:CorrelativeResultsfromAZD9496OralSERDPhaseITrial.ClinCancerRes40 2018;24(23):5860-72.41 30. JanssonS,BendahlPO,LarssonAM,AaltonenKE,RydenL.Prognosticimpact42 ofcirculatingtumorcellapoptosisandclustersinserialbloodsamplesfrompatients43 withmetastaticbreast cancer inaprospectiveobservational cohort.BMCCancer44 2016;16(433.45 31. LarssonAM, Jansson S,Bendahl PO, LevinTykjaer Jorgensen C, LomanN,46 GraffmanC, et al. Longitudinal enumerationandclusterevaluationof circulating47

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FIGURELEGENDS1 2 Figure1.AggregationiscompromisedinMCF-7cellsincubatedwithpaclitaxel3

orvinorelbine4

aMCF-7cellsincubatedornot(untreated)withPaclitaxel(100nM)orVinorelbine5

(20nM)were seeded in 96-well low-attachment plates andmonitored by video-6

microscopy for 5 hours (adapted from [15]). Representative transmitted light7

microscopy imagesofcellaggregationat the indicatedtimepoints.Segmentation8

(red line) was performed using a dedicated MATLAB software. Blue lines9

correspond to isolated cells.b, cAnautomated imageprocessingprocedurewas10

usedtomeasuretheaggregateareaduringtheassayinthepresenceofpaclitaxel11

(a)orvinorelbine(b)andthepercentageofcompactionwascalculated fromthe12

normalizedareavariationrelativetotheinitialtimepoint.Datacorrespondtothe13

mean±SDof48aggregates/conditionfrom3independentexperiments.**,P<0.01;14

****,P<0.0001(MannWhitneynon-parametrictest).15

16

Figure 2. Anchorage-independent aggregation is inhibited in metaphase-17

blockedMCF-7breastcancercells.18

a Control (untreated) andmetaphase-synchronized (incubationwith nocodazole19

andMG132,seeMethodsandAdditionalfile1:Fig.S1)MCF-7cellswereseededin20

96-well low-attachment plates andmonitored by video-microscopy for 5 hours.21

Representative transmitted light microscopy images of cell aggregation at the22

indicated time points. Segmentation (red line)was performedusing a dedicated23

MATLAB software. Green lines correspond to the excluded holes, and blue to24

isolatedcells.bUsingtheautomatedimageprocessingdata,theaggregateareawas25

25

measured over time. The graph corresponds to the percentage of compaction1

calculatedfromthenormalizedareavariationrelativetotheinitialtimepoint.Data2

correspond to the mean ± SD of 48 aggregates for each condition from 33

independentexperiments.*,P<0.001(Mann-Whitneynon-parametrictest).4

5

Figure 3.Metaphase-blockedMCF-7 breast cancer cells form less cohesive6

clusters.7

a Aggregate circularity was determined in control (untreated) and metaphase-8

synchronized/blockedMCF-7cellsafter5hours.***,P<0.001(Mann-Whitneynon-9

parametric test). b Cluster cohesion analysis using a flush-assay. After 5 hours,10

clustersweremechanicallydissociatedbyrepeatedlyflushingwithamicropipette.11

Thenumberofdissociatedcellswasdetermined.Dataarethemean±SDof12wells12

foreachconditionfrom4independentexperiments.*,P<0.05;***,P<0.001(two-13

wayANOVA).14

15

Figure4.MonitoringMCF-7cancercellclusteringatthesingle-cellscale.16

MCF-7cellsthatexpresstheLifeAct-mCherryfluorescentreporterwereseededin17

PDMSmicro-wells placed in culture dishes (Additional file 2: Fig. S2) and their18

clusteringwasmonitoredbyvideo-microscopy.19

aRepresentativefluorescenceimagesofcontrol(untreated)MCF-7cellsatdifferent20

time-pointsduringclustering.Thedottedlineshowsthemicro-welledge.Scalebar:21

50µm. b Representative micrographs of a MCF-7 cell that express the LifeAct-22

mCherry fluorescent reporter during aggregation (left panels). Membrane23

protrusionswereautomatedlydetected(redline).Timeisindicatedinmin.Scale24

26

bar:10µm.(Rightpanel)Plotoftheaspectratioofasinglecellwithmeanvalue(µ)1

andstandarddeviation(s)over1hour.2

3

Figure5.Membraneprotrusionsrequireafunctionalactincytoskeleton.4

a Representative micrographs of MCF-7 cells that express the LifeAct-mCherry5

fluorescentreporterandincubatedornot(untreated)withCK666orlatrunculinA6

(LAT) at different time-points during aggregation. Cell contours andmembrane7

protrusions were automatedly detected (white line). Scale bar: 10µm. b8

Determinationoftheaverage(µ)andstandarddeviation(s)aspectratioofsingle9

cellsincontrol(untreated;UNT),andincellsincubatedwithCK666orlatrunculin10

A(LAT).*,P<0.05;**,P<0.01;****,P<0.0001(MannWhitneynon-parametrictest).11

Eachdotcorrespondstothevaluesofonecell.cGraphsshowingtheaspectratio12

andcircularityofthelargerclusters(seemicrographsinleftpanels,largerclusters13

areinred)incontrol(UNT)andCK666orlatrunculin(LAT)-treatedcells after314

hoursofclustering.Eachdotcorrespondstothevaluesofonewell.Forbandc,data15

are from5 independent experiments, and bars correspond to themean±SD. *, P16

<0.05;**,P<0.01;****,P<0.0001(MannWhitneynon-parametrictest).17

18

Figure 6. Metaphase-blocked MCF-7 cells do not form actin-dependent19

protrusionsduringanchorage-independentaggregation.20

aRepresentativefluorescenceimagesofmetaphase-synchronized/blockedMCF-721

cellsatdifferenttime-pointsduringclustering.Thedottedlineshowsthemicro-well22

edge. Scalebar:50µm.bLeft: Imagesof aPDMSmicro-wellwithuntreatedcells23

(top)andmetaphase-synchronized/blockedMCF-7cells(Met-sync,bottom)atthe24

27

lasttimepointofthetime-lapseexperiment(180min).Right:Correspondingbinary1

imagesattheendoftheimagesegmentationprocess.Circles,edgeofthemicro-well;2

red, the largest cluster formed thatwasused for theaspect ratioandcircularity3

analysis.c Graphs showing the aspect ratio (left) and circularity (right) analysis4

results for the larger clusters in control (untreated; UNT) and metaphase-5

synchronized(Met-sync)cellsafter3hoursofclustering.Eachdotcorrespondsto6

thevaluesinonemicro-wellfrom5independentexperimentsandbarscorrespond7

to themean±SD. *,P<0.05; **,P<0.01 (MannWhitneynon-parametric test).d8

Representative micrographs of control (untreated) and metaphase-9

synchronized/blocked (Met-sync) MCF-7 cells that express the LifeAct-mCherry10

fluorescent reporter during aggregation (Left panels). Thewhite line shows the11

regionofinterest(ROI)usedfortheaspectratiodetermination.eGraphsshowing12

the average (µ) and standard deviation (s) aspect ratio of control (UNT) and in13

metaphase-synchronized/blockedcells(Met-sync)after1hourofaggregation.Each14

dotcorrespondstoonecellandthebarscorrespondtothemean±SD.Dataarefrom15

5independentexperimentswith5-6cellsanalyzedperexperiment.****,P<0.000116

(MannWhitneynon-parametrictest).17

18

19

20

21

22

23

24

28

ADDITIONALFILES1 2

Additionalfile1:3

SupplementaryFigureS1.Synchronizationprocedure4

aSchematicrepresentationofthesynchronizationprocedure.Cellswereincubated5

withnocodazolefor20h,andMG132wasaddedtothemediumforthelast30min.6

Thereafter,theculturemediumwasreplacedbymediumcontainingonlyMG132for7

1.5hbeforemitoticshake-offandstartoftheaggregationassayofcellsarrestedin8

mitosistomonitortheirclustering.9

b Flow cytometry analysis of control (untreated) and mitosis-arrested cells10

(nocodazole/MG132). Mitotic cells were detected with the mitotic-specific11

monoclonal3-12-I-22antibody.12

cRepresentativefluorescencemicroscopyimages(DAPIandalpha-tubulin)ofcells13

blocked inmitosis(nocodazole/MG132)andusedfor theclusteringassays.Scale14

bar:10µm.15

16

Additionalfile2:17

SupplementaryFigure S2.Microdevice to study clustering at the single-cell18

scale19

aMaskusedforthefabricationofthesiliconwafer.bOnearrayof9PDMSmicro-20

wells(outerdiameter:650µm,innerdiameter:450µm,andheight:200µm)thatare21

(c)gluedonthebottomofthecompartmentsofCELLviewTMcellculturedishesfor22

monitoringbytime-lapsevideo-microscopy.23

Additionalfile3:24

29

SupplementaryMovie1.Cellclusteringinthemicrodevice1

Time-lapse image acquisition of MCF-7 cells that express the LifeAct-mCherry2

fluorescent reporter during clustering. Transmitted and mCherry fluorescence3

imagesaremerged.Movieduration:3hours.Scalebar:50µm.4

5

Additionalfile4:6

Supplementary Movie 2: Kinetics of one MCF-7 cell during clustering in a7

PDMSmicro-well.8

Fluorescence images from time-lapse acquisition of one controlMCF-7 cell that9

expresses the LifeAct-mCherry fusion protein. On the right panel, thewhite line10

correspondstotheROIusedformorphometricparameterdetermination.Scalebar:11

10µm.12

13

Additionalfile5:14

SupplementaryMovie3:CellkineticsofoneMCF-7cellincubatedwithCK66615

duringclusteringinaPDMSmicro-well.16

Fluorescenceimagesfromtime-lapsemonitoringofoneCK666-treatedMCF-7cell17

thatexpressestheLifeAct-mCherryfusionprotein.Ontherightpanel,thewhiteline18

correspondstotheROIusedformorphometricparameterdetermination.Scalebar:19

10µm.20

21

Additionalfile6:22

SupplementaryMovie4:CellkineticsofonelatrunculinA-treatedMCF-7cell23

duringclusteringinaPDMSmicro-well.24

30

Fluorescenceimagesfromtime-lapsemonitoringofonelatrunculinA-treatedMCF-1

7 cell that expresses the LifeAct-mCherry fusion protein. On the right panel, the2

whitelinecorrespondstotheROIusedformorphometricparameterdetermination.3

Scalebar:10µm.4

5

Additionalfile7:6

Supplementary Figure S3. Characterization of clusters in control and7

experimentalconditions.8

a,bGraphsshowingtheaspectratio(a)andcircularity(b)analysisresultsforthe9

largerclusters formed inmicro-wells inMCF-7cellsincubatedornot(untreated,10

UNT) with nocodazole and MG132 (i.e., metaphase-synchronized/blocked, Met-11

sync),orwithMG132(MG)ornocodazole(Noco)aloneafter3hoursofclustering.12

Each dot corresponds to the values in one micro-well from 5 independent13

experiments and bars correspond to the mean ± SD. c,d Determination of the14

average(µ)(c) and standarddeviation(s)(d)aspectratio insingleMCF-7cells15

incubatedornot (untreated,UNT)withnocodazoleandMG132 (i.e.,metaphase-16

synchronized/blocked,Met-sync),orwithMG132(MG)ornocodazole(Noco)alone17

after 1 hour of aggregation. Each dot corresponds to one cell and the bars18

correspond to themean±SD.Dataare from5 independentexperimentswith5-619

cellsanalyzedperexperiment.*,P<0.05;**P,<0.01;****,P<0.0001(MannWhitney20

non-parametrictest).21

22