Role of near-infrared fluorescence imaging in the resection of metastatic lymph nodes in an optimized orthotopic animal model of HNSCC

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resection of metastatic lymph nodes in an optimized

orthotopic animal model of HNSCC

I. Atallah, C. Milet, R. Quatre, M. Henry, E. Reyt, J.-L. Coll, A. Hurbin,

C.A. Righini

To cite this version:

I. Atallah, C. Milet, R. Quatre, M. Henry, E. Reyt, et al.. Role of near-infrared fluorescence imaging

in the resection of metastatic lymph nodes in an optimized orthotopic animal model of HNSCC.

European Annals of Otorhinolaryngology, Head and Neck Diseases, Elsevier Masson, 2015, 132 (6),

pp.337-342. �10.1016/j.anorl.2015.08.022�. �hal-02349440�

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Availableonlineat

ScienceDirect

www.sciencedirect.com

Original article

Role of near-infrared ﬂuorescence imaging in the resection of

metastatic lymph nodes in an optimized orthotopic animal model of

HNSCC

I. Atallah

^a^,∗,^b^,^c

, C. Milet

^b^,^c

, R. Quatre

^a^,^b^,^c

, M. Henry

^b^,^c

, E. Reyt

^a^,^b

, J.-L. Coll

^b^,^c

,

A. Hurbin

^b,c,1

, C.A. Righini

^a,b,c,1

aCliniqueuniversitaired’ORL,CHUdeGrenoble,BP217,38043Grenoblecedex9,France

bUniversitéJoseph-Fourier,BP53,38041Grenoblecedex9,France

cUnitéInsermU823,institutAlbert-Bonniot,BP170,38042Grenoblecedex9,France

a r t i c l e i n f o

Keywords:

Near-infraredﬂuorescenceimaging-guided surgery

Headandnecksquamouscellcarcinoma Metastaticadenopathy

˛vˇ3integrin RAFT-c(RGD)4

a b s t r a c t

Objectives: Tostudytheroleofnear-infraredﬂuorescenceimaginginthedetectionandresectionof metastaticcervicallymphnodesinheadandneckcancer.

Materialsandmethods:CAL33headandneckcancercellsofhumanoriginwereimplantedintheoral cavityofnudemice.Themicewerefollowedupaftertumorresectiontodetectthedevelopmentof lymphnodemetastases.Aspecificfluorescenttracerfor˛vˇ3integrinexpressedbyCAL33cellswas injectedintravenouslyinthesurvivingmicebetweenthesecondandthefourthmonthfollowingtumor resection.Anear-infraredfluorescence-imagingcamerawasusedtodetecttraceruptakeinmetastatic cervicallymphnodes,toguideoflymph-noderesectionforhistologicalanalysis.

Results: Lymphnodemetastaseswereobservedin42.8%ofsurvivingmicebetweenthesecondand thefourthmonthfollowingorthotopictumorresection.Near-infraredﬂuorescenceimagingprovided real-timeintraoperativedetectionofclinicalandsubclinicallymphnodemetastases.Theseresultswere conﬁrmedhistologically.

Conclusion:Nearinfraredﬂuorescenceimagingprovidesreal-timecontrastbetweennormalandmalig- nanttissue,allowingintraoperativedetectionofmetastaticlymphnodes.Thispreclinicalstageisessential beforetestingthetechniqueinhumans.

1. Introduction

Inheadandnecksquamouscellcarcinoma(HNSCC),surgery shouldachievecompleteresectionwithadequatesafemargins.In additiontotumorresection,giventherateoflymph-nodeinva- sioninHNSCC,which canbeashighas30%to40%in patients withoutanyclinicaladenopathy(cN0),neckdissectionshouldin principlebeassociated,forbothdiagnosticandtherapeuticpur- poses.Itshouldbeascompleteaspossibleandshouldincludeall thelymph-nodeareasinthelymphaticdrainageterritoryofthe primarytumor.Intheliteraturetherearefewtechniquesallowing real-timeintraoperativedetectionofmetastaticlymphnodes.Such

∗ Correspondingauthor.

E-mailaddress:[email protected](I.Atallah).

1Thetwoauthorscontributedequallytothestudy.

techniquesshouldoptimizeneckdissectionbymakingitselective, withonlymetastaticadenopathiesbeingresected.

Near-infraredfluorescenceimagingfordiagnosis,treatmentand follow-upofHNSCCisafastdevelopingfield.Itprovidesreal-time informationonthelocalizationandextensionofmalignanttissue throughcreationofaspecificcontrastbetweennormalandcancer tissue.Itrequiresfluorescentprobesthatspecificallytargetcancer cells.Oneofthemostimportanttargetsis˛vß3integrin,whichis widelyexpressedonthesurfaceoftumor-vesselendothelialcells andbymosttumorcells,includingHNSCC,duringmigrationorby cellslocatedoninvasionfrontlines[1–3].Previousstudiesshowed thatnear-infraredfluorescenceimaging targeting␣vß3integrin providesbenefitinHNSCCsurgerybyimprovingresectionquality throughthedetectionoftumorresiduewithinthesurgicalbedthat wouldbeinvisibletothenakedeyeevenundermagnifyinglenses, sothattheresiduewouldbeoverlookedinpurelymacroscopically guidedresection.Itwasfurtherdemonstratedthatthisdetectionof cancerresiduespositivelyimpactedrecurrence-freesurvivalinan http://dx.doi.org/10.1016/j.anorl.2015.08.022

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2 I.Atallahetal./EuropeanAnnalsofOtorhinolaryngology,HeadandNeckdiseasesxxx(2015)xxx–xxx

orthotopicanimalmodelofHNSCC,whichwasdevelopedbyour team[4,5].

The present study examined the role of near-infrared ﬂuo- rescence imaging in the detection and resection of metastatic cervicaladenopathiesinourorthotopicanimalmodelofHNSCC.

We injected a fluorescentpeptide specificfor ␣vß3 integrinin ordertotargetcancercellswithinadenopathies.Thistargetingwas visualizedbyanear-infraredfluorescence-imagingdevicethatwas previouslyminiaturizedandspecificallyadaptedforfutureappli- cationsinHNSCCsurgery.

2. Materialandmethods 2.1. Cellline

Inourexperiments,weusedtheCAL33HNSCCcellline,supplied freeofchargebytheAntoineLacassagneCancerCenteroncophar- macologylaboratory,Nice(France).Itwasestablishedfromatissue sample taken from a human oral cavity moderatelydifferenti- atedmalpighiancarcinoma[6],andshowedstableexpressionof luciferasegenebylentiviraltransfection,allowinginvivobiolu- minescenceimagingtodetectlymph-nodemetastases.Cellswere keptinDulbecco’smodiﬁedEagle’smedium(DMEM)with10%fetal calfseruminahumidincubatorat37^◦Candanatmospherecon- taining5%CO₂.

2.2. Moleculartargeting

AngioStamp^TM 800(Fluoptics, Grenoble,France) is apeptide coupledtoaﬂuorophore.Itshowsspeciﬁcbindingto˛vˇ3integrin.

It has a maximal absorbance at 781 nm and maximal emission at 794 nm. It was injected intravenously into the mouse tailunderaninhaledgeneralanesthesia, at10 nmolpermouse 16–24hoursbeforefluorescenceacquisition.Theinvivofluores- centsignalwasacquiredbythenear-infraredfluorescenceimaging systemdescribedbelow.

2.3. Near-infraredﬂuorescenceimagingsystem

FluoStick^TM (Fluoptics,Grenoble, France) isa small-diameter imagingsystem,miniaturizedandspecificallyadaptedforfuture applicationsinHNSCCsurgery[5].Itisintendedforintraoperative fluorescenceimagingusingfluorophoreswithmaximalabsorbance around770nmandmaximalemissionaround820nm.

2.4. OrthotopicanimalHNSCCmodel

Theanimalsusedin thestudywerefemale nudemiceaged 5–6weeks,fromJanvierLabs(LeGenest,Saint-Isle,France).The experimentalprotocolwasapprovedbytheFrenchEducationand ResearchMinistry(experimentationauthorizationno.00392.02).

The orthotopic HNSCC model was previously described and validated[4,5].Brieﬂy,a0.5–1mmtumorfragmentfromtumor developingsecondarilytosubcutaneousCAL33cellimplantation inadultmicewasimplantedintheinneraspectoftheanimal’s cheek(n=14).Oncedeveloped,thetumorwascompletelyresected throughanexternalincisionofthecheek,undernear-infraredﬂo- rescenceimagingafterintravenousinjectionofAngioStamp^TM800.

Micewerefollowedupfor6monthspost-explantationtodetect latetumorrecurrenceandlymph-nodemetastasisthroughbiolu- minescenceimagingwhichisdescribedbelow,thensacriﬁcedat endoffollow-uporonsetoftumorrecurrenceorclinicalorsubclini- callymph-nodemetastasis;weight-lossexceeding15%andgeneral healthdeteriorationalsoledtosacriﬁce.

2.5. Bioluminescenceimaging

In vivo bioluminescence imaging was used to track tumor growth and detect recurrence and/or metastatic adenopathies.

Fiveminutesbeforeacquisition,intraperitonealluciferininjection (150mg/kg)was performedand thenanimals werekeptunder generalinhaledanesthesia(isoﬂurane4%forinductionand1.5%

formaintenance)duringbioluminescenceacquisitionontheIVIS Kineticsystem(CaliperLifeSciences).

2.6. Lymph-nodesurgery

Whenmetastaticadenopathywasdetected,AngioStamp^TM800 wasinjectedintravenouslyat10nmolperinjection.Micewere anesthetizedwithisoflurane4%forinductionand1.5%formaintenance;16–24hours after injection, a fluorescence acquisition wastakenbeforeandafterwidecervicalincisionexposingallthe anatomicstructuresoftheneck.Micewerethensacrificedbycer- vicaldislocation.Suspectadenopathiesandadenopathiesshowing fluorescentsignalwereresectedandanalyzedhistologically.Con- trollymphnodes(unsuspicious,non-fluorescent)werealsoexcised andanalyzed.

2.7. Hematoxylin-eosin(H&E)staining

Lymph-nodetumorcelldetectionwasperformedonhistologic sliceswithH&Estaining.Sampleswerefixedin4%PFA,dehydrated insuccessivelyincreasingalcoholbaths,thenincludedinparaffin andslicedbymicrotomewith7␮mthickness.Slicesweredeparaf- finized in xylene baths, rehydrated in successively decreasing alcoholbaths, thenstainedwithhematoxylinfor 4minutesand rinsedinflowingwater,stainedwitheosinfor2minutesandrinsed againinflowingwater.Thestainedsliceswerefixedin100%ethanol thenxylene,andslidemountedforhistologyunderaBX41Olympus microscope.

3. Results

3.1. Developmentofanorthotopicmodeloflymph-node metastasisinHNSCC

Fourteen mice with orthotopic HNSCC underwent complete near-infraredfluorescence-guided resectionand were then fol- lowed up for 6 months on bioluminescenceimaging todetect tumorrecurrenceormetastaticadenopathy.Fourmice(28.5%)had localrecurrenceduringthefirst2months.Duringthefollowing 4 months, 3 miceshowed signs of deteriorated general health status with>15%weightloss, withoutrecurrenceor detectable metastatic adenopathy, and were sacrificed. Bioluminescence detectedmetastatic adenopathy(Fig. 1)in 3of the7 surviving mice(42.8%)betweenthe3rdand5thmonthpost-implantation (2ndto4thmonthafterorthotopictumorexplantation).Whenthe adenopathieswerelarge,theywerealsodetectablemacroscopi- callyandonpalpation.Histologicanalysisofresectedsuspicious lymphnodesfoundmetastasisofmalpighiancarcinoma(Fig.2A).

3.2. Near-infraredﬂuorescenceimagingguidedlymph-node surgeryinHNSCC

Thedetectedmetastaticadenopathiesweresampledbynear- infrared fluorescence-guided surgery after AngioStamp^TM 800 injection. In the absence of clinically detectable metastatic adenopathy,preoperativebioluminescenceimagingandpre-and intra-operativefluorescenceimagingshowednocervicalfluores- centsignal(Fig.3A).Histologicanalysisofasampledcontrollymph nodefoundnoabnormality(Fig.3A,yellowarrow).Conversely,

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Fig.1.Detectionofmetastaticadenopathies.Metastaticlymphnodescouldbeeitherdetectedonbioluminescenceimagingormacroscopically(A).Insomecases,metastatic lymphnodesareonlydetectedbybioluminescenceimaging(B).

Fig.2. Histologic(H&E)lymph-nodeanalysis.A.Inmetastaticadenopathy,squamouscellcarcinoma(dottedarrow)inﬁltratesalmosttheentirelymphnode;notealsothe interface(star)betweenresiduallymphocytepopulation(arrow)andmetastasis.B.Controllymphnode,showingastructurefreeofanytumorinvasion.

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Figure3.Contributionofnear-infraredfluorescenceimaginginHNSCClymph-nodesurgery.MouseApresentsnodetectablemetastaticadenopathyonmacroscopicexam- ination,bioluminescenceimagingorfluorescenceimaging.MouseBpresentsametastaticadenopathymacroscopicallydetectableaswellasonpre-andintra-operative bioluminescenceandfluorescenceimaging;pathologicanalysisconfirmedthemetastaticcharacteroftheadenopathy(redarrow).MouseCpresentsacervicalbioluminescent signal,butnoadenopathyisdetectablepreoperatively.Intra-operatively,ahypertrophicadenopathy(bluearrow)showsnofluorescentsignal;incontrast,anunsuspicious lymphnodedoesshowafluorescentsignal(greenarrow).Bothadenopathiesweresampledforhistologyandcomparedwithacontrollymphnode.Thefluorescentadenopa- thyshowedmetastasisofsquamouscellcarcinoma,whilethemacroscopicallysuspiciousnon-fluorescentadenopathyandcontrollymphnodeshowednoabnormalityon histologicanalysis.

in case of macroscopically detectable preoperative adenopathy (Fig.3B, redarrow)associatedwithabioluminescencesignal,a fluorescentsignalwasalsoobservedonpre-andintra-operative fluorescenceimaging(Fig.3B, redarrow).Histologic analysisof thehypertrophiedfluorescentlymphnodedemonstratedmetas- tasis (Fig. 3B, red arrow). Finally, in case of adenopathy with

preoperative bioluminescencesignalbut macroscopically unde- tectableduetolackofmorphologicalsignsofmalignancy(Fig.3C), asignalwasobservedonlyonintraoperativeﬂuorescenceimaging (Fig.3C,greenarrow).Thislymphnodedidnotseemsuspiciouson intraoperativemacroscopicexamination,unlikeanotherhypertro- phiedbut non-ﬂuorescentnode(Fig.3C,bluearrow).Histologic

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analysis showed the ﬂuorescent adenopathy to be metastatic (Fig.3C,greenarrow)andthehypertrophiednon-ﬂuorescentnode, likeacontrolnode,tobenormal(Fig.3C,blueandyellowarrows).

Theresultsofnear-infraredﬂuorescenceimaging-guidedlymph- nodesurgerydemonstratedthatﬂuorescenceimagingeffectively guidedintraoperativesamplingofmetastaticlymphnodes,even whenclinicallyundetectable.

4. Discussion

Developmentandassessmentofnewdiagnosticandtherapeu- tictoolsforHNSCCrequirepriortestinginasuitableanimalmodel, whichneedstobereproducibleandrepresentativeofHNSCC.Ina previousstudy,ourteamdevelopedanorthotopicanimalmodelof HNSCC,with30days’survivalwithinsitutumor.Weoptedfororal cavityimplantationoftumorfragmentsratherthandirectlyinject- ingtumorcells[5].Inotherorthotopicmodelsdescribedinthe literature,cellinjectionmayinducedisseminationofcancercellsby thehydrostaticpressurefromthesyringeduringinjection,result- ingintherapiddevelopmentoflargetumors,requiringsacrifice andthusmakingitdifficultforlymph-nodemetastasistodevelop [7–9].IncertainorthotopicanimalHNSCCmodelsusingcellinjec- tion,thecervical lymph-nodemetastasesthat diddevelopmay havebeenduetocelldisseminationbyhydrostaticpressureinthe lymphvesselsduringinjectionratherthantocellmigrationfrom theprimarysite[9,10].Suchmodelsthusfailtoreproducethereal- ityofthelymph-nodemetastasisprocess.Thepresentorthotopic animalmodelofHNSCCallowedtumorresectionwithoutsacrifice, andthuslongfollow-uptodetecttumorrecurrenceandthedevel- opmentofmetastaticadenopathy.Thepresentstudydemonstrated thatthemodelallowedmetastaticadenopathytodevelopin42.8%

ofmicebetweenthe2ndand4thmonthaftertumorresection,a percentageapproximatingthe30–40%rateoflymph-nodeinvasion inhumanHNSCC.Themodelthusfaithfullyrepresentsallcharac- teristicsofHNSCC,enablingtheimpactofnewsurgicalormedical techniquesontheprimarysiteandonmetastaticadenopathytobe assessed.

Several strategies have been suggested for fluorescence imaging-guidedHNSCCsurgery.Clinicalapplicationrequiresphar- macokinetic study of each newfluorescent probe. Indocyanine green(ICG)wasoneofthefirstfluorophorestestedinnear-infrared fluorescence-guidedHNSCCsurgeryandprovedeffectiveindetec- tingsentinelnodes.It showspassivedrainagebythelymphatic system.Ratherthanspecificallytargetingcancertissue,ICGistaken upbytheenhanced permeabilityandretention(EPR)effect[7], inwhichthelargediameterofendothelialfenestration,hemody- namic changes withintumoral neovessels and thelow level of lymphaticdrainageoftumorsleadstoanaccumulationofparti- clesandsmallagentsinthetumortissue.ICGisthusnotuseful foroptimizingHNSCCresectionmarginsordetectinglymph-node metastasis[11–13].Fluorescentprobesspecificallytargetingrecep- torsexpressedbyHNSCC,incontrast,providereal-timedistinction betweencancerousandhealthytissue.

Oneofthemostimportanttargetsoffluorescentprobesinoncol- ogyisavß3integrin,whichshowslargeexpressioninneovessels and certain tumors, includingHNSCC [1–3].It playsan impor- tant role in angiogenesis, cell proliferation and migration and metastasis.Amongprobestargetingavß3,theRAFT-c(-RGDfK-)₄ (regioselectively addressable functionalized template-arginine- glycine-asparticacid)peptideshowedspecificityinvitroandinvivo andhasbeencoupledtomanyfluorophorestocreatespecificflu- orescentprobessuchasAngioStamp^TM800,usedinthepresent study[14–16].

The present study confirmed previous findings in the opti- mization of tumor resection under near-infrared fluorescence

imaging.Thelocalrecurrenceratewas28.5%intheﬁrst2months afterresection, inagreementwith ourpreviousrateof 25±5%

[5]. AngioStamp^TM 800 was alsoinjected intravenously in sur- vivingmicebetweenthe2ndand 4thmonthpost-explantation.

Near-infrared fluorescenceimaging detectedclinically observed adenopathies but also those undetectable macroscopically pre- or intra-operatively; their metastatic character was confirmed by histology. In mice with cervical adenopathies on clinical examination and/or with bioluminescent cervical signal, near- infraredfluorescenceimagingwasthusabletoconfirmmetastatic status. Intra-operatively, it also helps the surgeon remove a targetedmetastaticadenopathythatisundetectablepre-orintra- operatively. Real-time near-infrared fluorescence imaging thus providesrealbenefit,specificallyinHNSCC,guidingneckdissec- tionsoasexhaustivelytoresectmetastaticcervicaladenopathies andoptimizeneckdissection.

5. Conclusion

Near-infraredﬂuorescenceimagingisapromisingandprecious toolinHNSCCsurgery.Aswellasimprovingtumorresectionqual- ityinthepresentorthotopicanimalmode,itguidedresectionof clinicallyunidentiﬁablemetastaticadenopathies.Thepresentpre- clinicalstudyisindispensablebeforehumantrials.

Disclosureofinterest

Theauthorsdeclarethattheyhavenoconﬂictsofinterestcon- cerningthisarticle.

Acknowledgments

ThankstotheGroupePasteurmutualitéfondationd’entreprise, theFrenchSocietyofORL(SFORL)andtheAvenirFoundationfor ﬁnancialsupport,andtoFluoptics(Grenoble,France)fortechnical help.

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