Research
article
Value
of
combining
dynamic
contrast
enhanced
ultrasound
and
optoacoustic
tomography
for
hypoxia
imaging
Anant
Shah
a,*
,
Nigel
Bush
a,
Gary
Box
b,
Suzanne
Eccles
b,
Jeffrey
Bamber
aaTheInstituteofCancerResearchandRoyalMarsdenNHSFoundationTrust,JointDepartmentofPhysicsandCRUKCancerImagingCentreintheDivisionof RadiotherapyandImaging–Sutton,UnitedKingdom
b
TheInstituteofCancerResearch,DivisionofCancerTherapeutics–Sutton,UnitedKingdom
ARTICLE INFO
Articlehistory:
Received28February2017
Receivedinrevisedform1August2017 Accepted8August2017
Keywords:
CEUS Microbubbles Optoacoustic Photoacoustic MSOT Registration
ABSTRACT
Optoacousticimaging(OAI)candetecthaemoglobinandassessitsoxygenation.However,thelackofa haemoglobinsignalneednotindicatealackofperfusion.Thisstudyusesanovelmethodtoassistthe co-registrationofoptoacousticimageswithdynamiccontrastenhancedultrasound(DCE-US)imagesto demonstrate, in preclinical tumour models, the value of combining haemoglobinimaging with a perfusionimagingmethod,showingthatalackofahaemoglobinsignaldoesnotnecessarilyindicatean absence of perfusion.DCE-USwas chosen forthis particular experiment becauseUS is extremely sensitive tomicrobubblecontrastagentsandbecausemicrobubbles,likeredbloodcellsbut unlike currently available opticalcontrastagents, donot extravasate.Significant spatialcorrelationswere revealedbetweentheDCE-USpropertiesandtumourblood-oxygensaturationandhaemoglobin,as estimatedusingOAI.ItisspeculatedthatDCE-USpropertiescouldbeappliedassurrogatebiomarkersfor hypoxiawhenplanningclinicalradiotherapyorchemotherapy.
©2017PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
1.Introduction
Hypoxiaisprevalentinmostmalignanthumancancersbecause thefunctionallyabnormaltumourvasculatureisnotabletomeet the excessive oxygen consumption needs of the proliferating tumour cells. This stimulates several compensatory biological processes,whichaltergeneexpressionprofiles[1,2]thatleadto metastasisandcancerprogression[3,4],andenableatumourto circumventchemo-[5]andradiotherapy[6].Ithasbeenwidely established that the hypoxic state of a tumour can influence treatmentoutcome[7–10].Inaddition,hypoxia-targetedtherapies areunderdevelopmentandinclinicaltrial[11].Therefore,fora numberofreasons,thereisanurgentneedforadiagnosticmethod for determining whethera tumour is hypoxic, so as toenable hypoxia-basedtreatmentindividualisation[11].
Commonly used methods for assessing tissue hypoxia in patientsareneedle electrode polarography,fordirect measure-ment of tumour oxygen saturation, and needle biopsies, for quantifying biomolecules expressed under hypoxic conditions
[11].Boththesetechniquesareinvasiveanddonotrepresentthe wholetumour,whichislikelytobecrucialforplanningtreatment.
Non-invasive imaging techniques for assessing hypoxia are therefore under development and investigation. However, trac-er-based hypoxia imaging methods such as phosphorescence quenching [12], electron paramagnetic resonance [13] and positron emission tomography[11] are of limited valuedue to restricted accessof the imaging probestothehypoxic regions. Otherimagingmodalities providewaystoinferhypoxia andits spatialandtemporalvariationbyquantifyingthehaemodynamics of tissue perfusion, total haemoglobin (HbT) or blood oxygen saturation(SaO2),eachwithitsownlimitations[11].
Quantitative SaO2 imaging is particularly challenging. For example, blood oxygen level dependent magnetic resonance imaging (BOLD MRI) is only sensitive to differences between twostatesoftheproportionofdeoxy-haemoglobin(Hb)present, ratherthananabsolutelevelofoxygenation.Intumourstudies,the changeofstateisachievedbychangingthelevelofoxygeninthe gasbreathed[14].Diffuseopticalspectroscopicimaging[15]isin principle abletoovercome this difficultybyusing recognisable propertiesofthewavelengthdependenceofopticalabsorptionin Hbandoxy-haemoglobin(HbO2)tomakeabsolutemeasurements ofHbandHbO2.However,itisaccompaniedbydepth-dependent low spatial resolution due to scattering of photons by tissue, leadingtodifficultyininterpretationofbloodSaO2orHbTvalues. This method works best when one can transmit light entirely
* Correspondingauthor.
E-mailaddress:Anant.Shah@icr.ac.uk(A.Shah).
http://dx.doi.org/10.1016/j.pacs.2017.08.001
2213-5979/©2017PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
ContentslistsavailableatScienceDirect
Photoacoustics
throughthebodyparttobeimagedandthereforeitsapplication haslargelybeenlimitedtobreastimaging[16–18].
Multispectraloptoacousticimagingalsousesthespectroscopic absorptionsignaturesofHbO2andHbtomakeabsolute measure-mentsofHbTandSaO2butisabletodothiswithahighspatial resolution(determined byacousticwave focusing)withoutthe problemsassociatedwithphotonscatteringandwithoutneeding totransmitlightentirelythroughthebody[19–21].The measure-mentsdo,however,dependonthesystem’ssensitivitytoidentify HbandHbO2,whichisaffectedbydepth-dependentsignaltonoise ratio,thespectraldependenceoflightattenuationinintervening tissueandthepresenceofotherchromophores(forexample oxy-and deoxy-myoglobin [22]). Additionally, “discrete sampling issues” and “imperfectnessin the reconstructionalgorithm”,as reportedbyDingetal.[23],couldalsoresultintheappearanceof ambiguous negative values that would make the spectral recognition of Hb and/or HbO2 more difficult. An absence of intra-tumouraloptoacoustichaemoglobinsignalisambiguousin thatitcannotbeknownwhethersuchregionsaretrulyavascularor whethertheyareperfusedbutwithatissuebloodvolumethatis belowthethresholdofdetection.
We hypothesise, therefore, that there would be value in combining multispectral optoacoustic imaging with a co-regis-tered sensitive perfusion imaging method for resolving such ambiguity.Inaddition,suchacombinativeimagingapproachmay be useful for gaining a more complete understanding of the hypoxic state of the tumour. For example, tumour vascular perfusioncharacteristicsmightonedaybecombinedwithlocal blood oxygenation estimates using an oxygen transport model
[24],toenableestimationofthelocalpartialpressureofoxygen withinthetumourcellenvironment.
To test this hypothesis we chose to combine multispectral optoacousticimagingwithmicrobubblebaseddynamiccontrast enhancedultrasound (DCE-US). It ispractical tocombinethese modalitiessincetheybothuseultrasoundandcanbeimplemented onthesame hardware.Moreover,microbubblescanbeused to imagethevasculatureatthemicroscopicscale[25,26]andtheir sizemakesthembehavelikeerythrocytes,effectivelypreventing theirextravasationfromthefenestratedmicrovasculature,making DCE-USsensitivesolelytothevascularperfusioncharacteristics, whereasalternativessuchascontrastenhanced(CE-)MSOT[27], DCE-MRI[28]orDCEcomputedtomography(CT)[28]usecontrast agents (indocyanine green, iodine or gadolinium, respectively)
[29] that movebetween the vascular and interstitial compart-ments.ThestudymightalsohavebeenperformedusingCE-MSOT, orevenDCE-MRIorDCE-CT,utilisingcontrastagentsthatwould remain intravascular, such as microbubbles loaded with dye molecules [30], gadolinium [31] and gold nanoparticles [32], respectively.Suchagents,however,remainintheresearchdomain andare notreadilyavailable, whereas microbubblesare conve-nientlyand commercially available, licensed for use in clinical ultrasound, and modern nonlinear signal processing makes ultrasoundscannersextremelysensitive totheirpresence(even detectingasinglemicrobubbleatadepthofmanycentimetresin tissue[26]).Theseprovided themainreasonswhyDCE-USwas chosenforthepresentstudy,knowingthatthefindingsshouldbe applicabletoothercontrastenhancedimagingmodalitiesgivena suitable contrast agent. A further advantage of studying the relationship between MSOT and DCE-US, rather than between MSOTandcontrastenhancedMSOT,MRIorCT,isthepotentialfor unanticipated discoveries which have useful consequences. Specifically,althoughitwasnotanaimofthisstudy,ifcorrelations betweenMSOTandDCE-USfeaturesweretobediscovered,this maysuggestawaytotranslatetheuseofvaluableMSOTimage informationintothe clinicinsituations, suchaswhen imaging deepwithinthebody,whereMSOTcannotbeemployed.
Although previous studies have compared DCE-US with multispectral optoacousticimaging in animal tumours [33–36], nonehaveinvestigatedtheabovehypothesisnorexploredthefull rangeofmicrobubble-basedperfusionpropertiessuchastimeof arrival,timetopeak,wash-intime,peakcontrast,areaunderthe curve(AUC),wash-inrateandwash-outratewithSaO2andHbT. Theaimsofthisstudyweretwofold:(i)toassesswhetherregions in optoacoustic images lacking an identifiable blood spectral signatureareindeedavascular,(ii)todeterminewhetherthereare relationships between the properties obtained using the two imagingapproaches.
AnMSOTinVision256-TFTM(iTheraMedical,Munich)wasused foroptoacousticimagingbecauseofitsreal-timefullspectraland wholemousecross-sectionalimagingability,andaclinicalscanner (AplioXGTM,Toshiba,Tokyo)wasusedforDCE-USsothatfindings fromthestudymayhavethepotentialfordirectclinicaltranslation and full advantage can be taken of commercially available microbubble agents, which as yet are not available for high-frequencyultrasoundimaging[37].Asaconsequence,thestudy requiredthedevelopmentofanovelexperimentalapparatusand scanningmethodtoassisttheprocessofco-registeringimagedata betweenthetwosystems,whichisalsodescribedinthispaper.
2.Methods
2.1.Celllines
Two pancreatic tumour models were chosen because they possess aheterogeneous vasculaturewithhypoxicregions [38– 40].The cellline, PDA-KPC-1GEMM,derivedfromKPCmice,a geneticallyengineeredpancreaticductaladenocarcinomamodel, wasobtainedfromtheUniversityofPennsylvania(Philadelphia, USA)[41].Thecarcinomacellline,MIAPaCa-2,wasobtainedfrom ATCC.Cells weregrown in Dulbecco's ModifiedEagle Medium, supplemented with 10% foetal bovine serum, in a humidified atmosphereof5%CO2inairat37C.
2.2.Tumourmodel
Groups of female CrTac:NCr-Fox1nu athymic nude mice,
approximately6weeksofage,wereinjectedsubcutaneouslyon therightflankwitheither3millionPDA-KPC-1-GEMMcells(n=2) or5 millionMIAPaCa-2cells (n=3).Theanimalswereimaged whenthetumourvolumereachedabout500mm3.
2.3.Optoacousticimaging
model-basedinversionalgorithm[42](ViewMSOTTMv3.6).The in-planeresolutionofsuchimageshasbeenmeasuredtobeofthe orderof 150
m
m(measuredbyiTheraMedical).Theanimalwas then translated along its long axisin stepsof 1mm to obtain additional cross-sectional images which together covered the wholetumour.Theslice-thicknessresolutionhasbeenmeasured tobeabout800m
m(measuredbyiTheraMedical).2.4.Ultrasoundimaging
Afteroptoacoustic imaging, for DCE-US imaging, the anaes-thetisedanimal in its holder was transferredundisturbed to a purpose-builtgantry,designedtoreplicatethewaythattheholder issupportedintheMSOTimagingsystem.AsshowninFig.1the gantryconsistedofawater-bagstandoffreplicatingthewatertank settingoftheanimalholderintheMSOTsystem.Theanimalinthe animal holder was submerged in the water warmed to a temperatureof34C.Maintainingtheanimalinitsoriginalholder, and submerged in water, restricted changes in posture and orientation of thetumour.The animal’s body temperaturewas maintainedduringtheimagingprocessusingaheatinglamp.A 1204BTlineararrayprobe(ToshibaAplioXGTMclinicalUSscanner) was mounted beneath the animal holder and water bag on a mechanicalstage,pointingupwardstoimagetheanimalin cross-section.Theultrasoundprobewastranslatedmechanicallyalong thelongaxisoftheanimalinstepsof1mmtocoverthewhole tumour.Afteraninitialfullmechanicalscantorecordthetumour’s echoanatomy,thetransducerwasalignedtothecross-sectionof thetumourhavingmaximumarea.100
m
LofSonazoidTM micro-bubbles[43]wastheninjectedthroughthecatheter.Interleaved non-linearcontrastmodeandfundamentalB-modeimageswere recordedataframerateof10Hzforupto40s.Non-linearcontrast modeimageswereobtainedbythescanner’sproprietary pulse-subtractioncodedharmonictechniquewithamechanicalindexof lessthan0.3andadynamicrangesettingof65dB.2.5.Dataanalysis
Priortooptoacousticimageanalysis,anynegativevaluepixels arisingasaresultofnoiseorreconstructionerrorswereassigneda
valueofzero.Undertheassumptionofnopositionorwavelength dependent fluence variation due to light attenuation, the reconstructeddatawerespectrallyunmixedusinglinear regres-sion(ViewMSOTTMv3.6)toresolvetheHbandHbO
2components in theoptoacousticimage. Foreverypixel in thereconstructed image,theunmixingalgorithmmodelstheobservedoptoacoustic spectrumasalinearcombinationoftheknownspectraofHband HbO2,tocomputeHbandHbO2imagesasthespatialdistribution of therelative concentrationof each absorber [21,44,45]. Mean SaO2andmeanHbTwerethencalculatedforuser-definedregions ofinterest(ROIs)withinthetumour(seebelow)usingtheEq.(1) [46,47]andEq.(2),
<SaO2>¼ cHbO2
E
HbT; *
ð1Þ
HbTE¼ cHbEþ cHbO2 E
; D D
D
ð2Þ
where cHbO2 E D
andDcHbEarethemeansovertheROIsofthe MSOTcalculatedconcentrationsofHbO2andHbrespectively.An “oxymap” image, showing the calculated SaO2 values for each pixel,wasalsoobtained,usingtheViewMSOTsoftware.Thiswas notusedforcalculatingthemeanSaO2valuesinROIsbecauseit wasobservedtobesubjecttopixeldropoutandthesoftwaredid not account for thezero valuepixels for calculating the mean valuesofSaO2inaregionofinterest.
TheMSOTandDCE-USimageshavingmaximumtumour cross-sectionalareawereassumedtocorrespondtoasimilarplaneand wereregisteredmanuallyas follows,utilisingrigid body trans-formationswithinMicrosoft’sExpressionEncoder4TM(Microsoft, Washington).First, thespectrally unmixedand oxymapimages werespatiallyrescaled,maintainingtheaspectratio,tomatchthe 10mmscalebartothatoftheDCE-USimage.Afterrescaling,the MSOT images were rotatedand translated sothat the tumour boundary and any major blood vessels observed in the single wavelengthimageat800nm(isosbestic pointofHb andHbO2) matched those of the non-contrast and contrast ultrasound images. Blood vessels were made more visible in the contrast mode ultrasound images by subtracting the pre- and post-microbubble injectionsequences (examples are shown later in Figs.4(E)and5(E),encircledbyaredboundary).Thespectrally unmixedandoxymapimagesweretranslatedandrotatedtothe samedegree.Rigidbody-transformationswereassumedsufficient forregisteringthetwoimagingmodalitiesbecausetheanimalwas maintainedinitsoriginalholderandsubmergedinwater,which restricted changes in animal posture and orientation of the tumour.
RegionsofthetumourshowinganabsenceofHbO2andHbon the spectrally unmixed optoacoustic images were selected by manual outlining to assess their perfusion characteristicswith DCE-US.Timeintensitycurves(TICs)werecalculatedasthemean contrastsignalwithineachROIversestime,afterbackgroundecho image subtraction, using a program written in Matlab (2010b, MathWorks,Natick,MA).
Inordertofurtherassessanypotential relationshipbetween HbTandSaO2valuesandDCE-USperfusioncharacteristics,ROIs were selectedin theperiphery and in thebody of the tumour wheretheoptoacousticimagesshowedapresenceofblood,and thesameregionswereselectedontheDCE-USimagesequences. SevenpropertieswerecalculatedfromtheTICsandcomparedwith SaO2 andHbT,ascalculated optoacoustically.The peakcontrast (maximumcontrastvalueattainedbytheTIC),timetopeak(time requiredforthemicrobubblestoreachthepeakcontrast)andthe
Fig.1.TheDCE-USimagingapparatuswhichreplicatesthearrangementusedto supporttheanimalholderintheMSOTinVision256TMandthusprovidesagood
timeofarrival(theintersectionpointbetweenthebaselineanda tangentofthewash-inrise)ofmicrobubblesfordifferentregions ofthetumourwereobtainedbyvisuallyfittingasmoothcurveto eachTIC.Thewash-intimewasobtainedasadifferencebetween the time to peak and time of arrival. The wash-in rate was determinedbymeasuringtheslopeofastraightlinebetweenthe peakcontrastandcontrastatthetimeofarrival.Thewash-outrate of microbubbles was calculated as the decay constant of an exponentialdecaycurvethatwasfittotheTICdatabetweenthe timeofpeakcontrastandthe40stimepoint.
To estimate andassesstherelationships ofSaO2and HbT to DCE-USproperties,twotypesofanalyseswerecarriedout.First,a within-tumouranalysiswasconductedinwhich,foreachtumour, thecorrelationbetweeneachoptoacousticandeachDCE-USimage property was evaluated. For each tumour and each pair of properties,themeanpropertyvaluewascalculatedineachROI, afterwhichthePearsoncorrelationcoefficientwascalculatedover thenumberofROIsinthattumour.Thisallowedtherelationships between the optoacoustic and the DCE-US properties to be assessedwithouttheconfoundingeffectofinter-tumourvariation. However, the significance of any such correlations, if present, would inevitably be low because the number of ROIs in each tumourwasnevermorethanseven,andwassometimesasfewas four.Thereforethesignificanceofthewithin-tumourcorrelation coefficientswasassessedbystudyingtheirconsistencyacrossallof thetumours,asrevealedbyaStudent’st-testappliedtothemeans andstandard deviations(acrosstumours) ofthewithin-tumour correlationcoefficientsforeachpairofproperties.
Second, thecorrelationbetweeneach optoacousticand each DCE-USproperty was evaluated using allROIs for alltumours. Here, the correlations were expected to be low, and of low significance,becauseoftheconfoundinginfluenceofinter-tumour variations.Therefore,thisanalysiswasrepeatedafterapplyingan approximatecorrection method, theobjective of which was to estimatethevaluesofthecorrelationcoefficientsthatwouldbe expectediftherewerenointer-tumourvariation.Thiscorrection methodbegan bycalculatingfor eachmousetheslope aMiand
intercept bMi in the least squares best fit of the linear model
(y=aMix+bMi)forthevariationofimagepropertyywithimage
propertyxoverthetumourROIsforthatmouse.Themeanslopea
and mean interceptb over allsuchfits for all micewerethen calculatedusing:
a¼N1X
N
i¼1
aMi b¼
1
N XN
i¼1
bMi; ð3Þ
whereNisthetotalnumberofmice.
Acorrecteddatapoint,correctedforthedepartureofagiven mouse’sbestfitlinearrelationshipfromtheaveragerelationship, wascomputedforeachdatapointcorrespondingtoeachROIusing Eq.(4):
yMi;kðcorrectedÞ¼yMi;kþðyyMiÞ; ð4Þ
wherey¼axkþbandyMi=aMixk+bMi,andkistheROInumberfor
the mouse. New correlation coefficients rcorrected, and their
correspondingpvalues,werecalculatedusingthesecorrecteddata points.
TheAUC,correlationcoefficientsandcorrectionsfor between-tumour variations were calculated using PrismTM version 7 (GraphPad, San Diego). Means and standard deviations, and statistical assessments using Student’s t-test, were calculated usingMicrosoftExcel12007TM.
3.Results
3.1.Optoacousticimaging
Forbothtumourmodels,theoptoacousticsignalsfoundatthe peripheryofthetumoursweretypicallystrongerthanthosefound inthetumourbody;examplesareshowninFigs.2Aand3,A.In ordertoallowthevariationsinimageappearancebetweenmiceto bebetterappreciated,imagesetsfromtwoadditionalmice(M2 andM4)areprovidedinFigs.S1andFig.S2,respectively,inthe supplementary section. Spectral unmixing revealed a strong
Fig.2.A)AreconstructedsinglewavelengthMSOTimageofmouseM1(exampleofaPDA-KPCtumour)at800nm.Thedashedyellowlineindicatesthetumour.B)The correspondingspectrallyunmixedimage,showingthedistributionofHbandHbO2componentsinblueandredrespectively.C)Theoxymapimage,showingthedistribution
oftheSaO2values.Theyellowscalebaratthelower-leftineachoftheseimagesis5mm.TheyellowarrowsinimagesBandCindicatesthecentreofaregionthatlacksany
spectralsignatureofblood.Thebackground,Hb,HbO2andoxymapcolourbarsreflectthemagnitudeofthephotoacousticsignals,deoxy-haemoglobin,oxy-haemoglobinand
SaO2,respectively,ascalculatedbytheMSOTsystem.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthis
presenceofHbO2intheperipheryofthetumours(Figs.2Band3B), correspondingtoahighSaO2value(Figs.2Cand3C).Thebodyof thetumourshadahigherproportionofHbincomparisontothe periphery(Figs.2Band3B),correspondingtoalowSaO2value.The PDA-KPCtumourshowedanabsenceofHb/HbO2signalinitscore, asevidencedbyblackregionsandmarkedbyarrowsinFig.2Band C. The MIA PaCa-2 tumour illustrated showed heterogenous vascularisationinitsbody,withanabsenceofbloodsignalinthree segments,asindicatedbyarrowsinFig.3C.
3.2.Contrastmodeimagingandregistrationwithoptoacoustic imaging
In ordertoassess whethertumour regions having noblood signalonoptoacousticimagingalsolackedobservableperfusionon DCE-US, ROIs were drawn around black regions on the opto-acousticHb:HbO2image,suchasthecoreofthePDA-KPCtumour in Fig. 4(D) (dashed green and dark green) and in the three differentsegmentsofthetumourMIAPaCa-2,asseeninFig.5(D) (top-leftdarkregion(greencolour),top-rightdarkregion(brown) andbottomdarkregion(pink)).ThesameROIswereappliedtothe DCE-US image sequences to obtainTICs (Figs. 4F and 5F) and evaluatetheperfusioncharacteristics.
Figs.4Cand5CshowthespectrallyunmixedMSOTimagesas transparentoverlaysontheDCE-USimagesofthesameexample PDA-KPC and MIAPaCa-2 tumours as shown in Figs. 2 and 3, respectively.Thecontrastmodeultrasoundimagesofthetumours beforeandaftermicrobubbleinjectionareshowninFig.4AandB, forthePDA-KPCtumourcase,andFig.5AandB,fortheMIAPaCa-2 tumourcase.
Inordertoassesswhetherthemicrobubbleperfusion proper-tieswerecorrelated ingeneral withSaO2 and HbTvalues, ROIs werealsoselectedinotherpartsofthetumourandinafeeding bloodvessel.ExampleROIsareillustratedinFig.4Dinred(blood vessel), dashed purple (upper rim), orange (upper body), and
dashedcyan(lowerrim),andFig.5Dinred(bloodvessel),dashed purple(upperrim),dashedyellow(innerbloodvessel),anddashed cyan(lowerrim).
Figs.4Eand5EshowtheoverlaysoftheselectedROIsonthe microbubble post-injection contrast-mode US images at time pointsof10,15and40s,aftersubtractionfromthepre-injection image.AsseeninFigs.4Eand5E,themicrobubblesarrivefirstat thefeedingbloodvesselatatimepointt10s,andsubsequently arriveinotherpartsofthetumour.AsseeninmouseM1(PDA-KPC) inFig.4,thetumourcore,lackedabloodsignalintheMSOTimage (Fig. 4D) and was echogenic on US before contrast injection (Fig.4A),wasperfusedonDCE-US,albeitwithalowbloodvolume andspeed(Fig.4E(t=40s)and4F,dashedgreencurve).Similarly, in mouse M3 (MIA PaCa-2) in Fig. 5, the three black regions (Fig.5D)werehighlyperfused,ascanbeseenfromthebackground subtractionimageatt=40s(Fig.5E)andTICsofthedarkregionsin
Fig.5F.Similarresultswereobservedinalltumourcases.These results clearly demonstrate the value of combining the two imagingmodalities,todifferentiatethepoorlyperfusedregionsof thetumourfromthosehavingnoperfusion,whichisdifficultto observewithMSOTalone.
3.3.Relationshipsbetweenmicrobubbledynamicpropertiesandblood contentandoxygenation
Forthewithin-tumouranalysis,asseeninFig.6AandC,there was aconsistentnegativecorrelationofSaO2andHbT withthe timeofarrival,timetopeakandwash-intime.Similarly,SaO2and HbT were positively correlated in almost all mice with peak-contrast,wash-out rate,wash-in rateand AUC,there beingone exception,a negativecorrelationofHbTwithAUCinmouseM1 (PDA-KPC).SincealimitednumberofROIs(4–7)wereselectedfor calculating these correlation coefficients, only a few were significant at the p=0.05 level (indicated by an asterisk in
Fig. 6A and C).However, significance is establishedbecause of
Fig.3.A)AreconstructedsinglewavelengthMSOTimageofmouseM3(exampleofMIAPaCa-2tumour)at800nm.Thedashedyellowlineindicatesthetumour.B)The correspondingspectrallyunmixedimage,showingthedistributionofHbandHbO2componentsinblueandredrespectively.C)Theoxymapimage,showingthedistribution oftheSaO2values.Theyellowscalebaratthelower-leftineachoftheseimagesis5mm.TheyellowarrowsinimagesBandCindicatesthecentreofaregionthatlacksany
spectralsignatureofblood.Thebackground,Hb,HbO2andoxymapcolourbarsreflectthemagnitudeofthephotoacousticsignals,deoxy-haemoglobin,oxy-haemoglobinand
SaO2,respectively,ascalculatedbytheMSOTsystem.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthis
theconsistencyofthewithin-tumourbehaviouracrossallofthe tumours; as shown in Fig. 6B and D, the mean correlation coefficientsweresignificantlydifferentfromzeroforallbutone optoacousticandDCE-UScombination.
Resultsforthecross-tumouranalysiswithoutaccountingfor theinter-tumouralvariationofthemeasurementsareshownin
Figs. 7–9. These results have been grouped soas to show the observed negative correlations in Fig. 7, positive correlations
involvingDCE-USbloodvolumemeasuresin Fig.8 andpositive correlationsinvolvingDCE-USratemeasuresinFig.9.
ReferringtoFig.7,significantnegativecorrelationwasobserved betweenSaO2 and DCE-USderived microbubbletime of arrival (r=0.5629,p=0.0015),timetopeak(r=0.597,p=0.0006)and wash-intime,(r=0.4966,p=0.0061).HbTalsoshowedanegative correlationwithtimeofarrival(r=0.4743,p=0.0093),time to peak (r=0.3625, p=0.0533) and wash-in time (r=0.239, p=0.2117). However, the correlation coefficients involving HbT wereconsistentlylowerthanthoseinvolvingSaO2,andwerenot significantfortimetopeakandwash-intime.
SignificantpositivecorrelationwasobservedbetweenHbTand theDCE-USbloodvolumeTICproperties(AUCandpeakcontrast). AsseeninFig.8,thecorrelationsbetweenHbTandAUC(r=0.4059,
p=0.0289)andpeakcontrast(r=0.4944,p=0.0064)werestronger and more significant than those involving SaO2 with AUC (r=0.3411, p=0.0702) and with peak contrast (r=0.3083, p=0.1038).
Significantpositivecorrelationswereobservedbetween micro-bubblewash-inrateandbothSaO2(r=0.5294,p=0.0031)andHbT (r=0.4313, p=0.0195).Although positive correlations werealso seen between microbubble wash-out rate and SaO2 (r=0.2854, p=0.1334)andHbT(r=0.3584,p=0.0563),neitherwassignificant atthep=0.05level.
AsummaryofthecorrelationcoefficientsfromFigs.7–9,and theirsignificance,isshowninFig.10.Thesignificancelevelsafter correctingforinter-tumouralvariations(Section2.5)areindicated asredasterisksinbracketsinFig.10.Thisapproximateattemptto
accountforinter-tumourvariabilityincreasedthesignificanceof7 ofthe14correlations(particularlythetimebasedTICproperties), had no effect on the significance of 5 and decreased the significancein2. Thescatterplotsoftherelationships between theoptoacousticimagingandthemicrobubbleDCE-US character-istics,aftercorrectingfortheinter-tumouralvariability(Figs.S3,S4 andS5)havebeenincludedinthesupplementarysection.
4.Discussion
To ourknowledge, this is the first time that a combinative imagingapproachofoptoacousticimagingandDCE-UShasbeen appliedtoanautomatedwhole-bodymousetomographic opto-acousticimagingsystem,theMSOTinVision256–TF,whichlacks thecapability toperform microbubblecontrastenhanced ultra-soundimaging.Forallfivetumours,itwasobservedthatcertain tumourregionsthat showednooptoacousticblood signalwere perfusedonDCE-US.Similarfindings canbeexpectedwith CE-MSOTusingacontrastagentthatdoesnotextravasate.Alowblood volume(asindicatedbyalowmicrobubbleuptakeonDCE-US), belowtheoptoacousticdetectionthresholdoftheimagingsystem, seemstobeaplausibleexplanation.Thesetumoursarenotprone tocontainconfoundingabsorbers;nordoesitseemlikelythatthe light attenuation in the subcutaneous tumours would be a contributing factor, that would obscure the recognition of haemoglobin.Nevertheless,toimprovethespatialhomogeneity and quantification of tumour SaO2 and HbT, incorporating an
algorithmtoestimatethewavelengthanddepth-dependentlight attenuation would be helpful. Tzoumas et al. [21] recently developed an approach to incorporate wavelength-dependent lightattenuationtoestimatebloodSaO2withindeeptissuewith more accuracy, using the MSOT system. Work in this area is underway.Analternative(hypothetical)explanationfortheabove observationisthatthemicrobubbles,giventheirsmallersizethan redbloodcells,werereachingareasofthetumournotaccessibleto thebloodcells.
TheSaO2valueswerefoundtobenegativelycorrelatedwith DCE-US time of arrival and time to peak with a high level of significance (Fig. 7).This implies that the intravascular micro-bubblesfollowthepathoftheredbloodcells.Astheoxygenated bloodtravelsfromthefeedingvesselstothemicrovasculatureof thetumour,thebloodSaO2shoulddecreaseonthepathtakenby the red blood cells. The extentof the decrease in SaO2 would dependonthemetabolicactivityanddemandforoxygen,which would be considerable for the periphery of the tumour in comparisonwithitshypoxiccore[48].Asimilarcorrelationwas also observed with HbT, although with a lower correlation coefficientandalowerlevelofsignificance.
HbT was positively correlated with AUC and peak contrast (Fig.8).ThesecorrelationswereexpectedasHbT,peakcontrast andAUCarealldependentonthelocalbloodvolume.Thisfinding broadlyconfirmstheresults(a)ofZionetal.[33],whoobserveda positive correlation of AUC with HbT in PC3 human prostate carcinomaxenograftmodels,and(b)ofEisenbreyetal.[36],who
Fig.6.(A)ThecorrelationcoefficientsforSaO2witheachDCE-UScharacteristicforindividualtumourcases(B)Themeansandstandarddeviationsoftheintra-tumour
had observed a positive correlationof peak contrastwith HbT (r=0.49,p=0.021).GiventhatZionetal.andEisenbreyetal.used different tumours, a different microbubble contrast agent and differentultrasound andoptoacousticimaging systemstothose employedhere, this confirmation of findings lendscredance to theirresultsandours,andincreasesconfidenceinotherfindings reportedonlyhere.
AsignificantpositivecorrelationofSaO2andHbTwithwashin rateofmicrobubbleswasobserved(Fig.9).Thewash-inandthe wash-outratesarerelatedtobloodvelocity.Theredbloodcells would havea higher velocityin theperipheral tumoural blood vesselsofferinglessresistancethanthetortuoustumour vascula-ture accompanied by a high interstitial pressure, commonly presentintheinteriorofthetumour[49].Regionsofthetumour showing a high SaO2 and HbT value, predominantly in the periphery,showed a higher wash-in rate in comparison to the regionshavinglow SaO2 andHbT values.Amoderatelypositive correlation of SaO2 and HbT with the wash-out rate was also observed,althoughitwasnotstatisticallysignificant.Thetransit time,thetotaltimeforthebubblestopassaparticularposition,
wasnotmeasuredbecausewedidnotallowtimeforthebubble contrastsignaltoreturntobaseline.Thissuggeststheneed for furtherworktoobservemicrobubblesforlongerperiods.However, thesumofwash-inrateandwash-outratecouldberegardedas directly related to the inverse of transit time, and a negative correlationbetweenSaO2andHbTwiththemicrobubbletransit timecanthereforebeexpected.
Thecorrelationsobservedbetweentheoptoacousticand DCE-US properties of different tumour regions suggest that a combinationoflowmicrobubblesignal,latemicrobubblearrival andslowmicrobubbletransitshouldbepredictiveoflowoxygen saturation.Thisremainstobetestedbutsuggestsnewrolesfor DCE-US in providing surrogate measuresof oxygenation levels, whichmaybeofvalueforexampleinpredictingradiationanddrug treatmenteffectivenessinclinicalsituationswhereatumourtobe treatedistoodeeptobeimagedoptoacoustically.
In ordertoassesswhetherthecorrelationcoefficients,along with their significance, may have been decreased by inter-tumouralvariationsinthefunctionalrelationshipbetweenimage properties,adifferencebetweentheoverallmeanrelationshipfor
Fig.7.VariationofSaO2(leftcolumn)andHbT(rightcolumn)withmicrobubbletimeofarrival(top),timetopeak(middle)andwash-intime(bottom).Correlationsbetween
SaO2andtheseDCE-USTICpropertieswerestrongerandmoresignificantthanthosebetweenHbTandtheDCE-USTICproperties.Thepvaluesindicatetheprobabilitiesthat
Fig.8.VariationofSaO2(leftcolumn)andHbT(rightcolumn)withmicrobubbleAUC(top)andpeakcontrast(bottom).CorrelationsinvolvingHbTwerestrongerandmore
significantthanthoseinvolvingSaO2.Thepvaluesindicatetheprobabilitiesthatthecorrelationcoefficients(r)arezero.Symbolsrepresentindividualmice: M1(PDA-KPC),
M2(PDA-KPC), M3(MIAPaCa-2),!M4(MIAPaCa-2), M5(MIAPaCa-2).Theindicativelinearregressionlineistoaidvisualinterpretationofthestrengthofcorrelation andisnotintendedtoimplythatalinearrelationshipexists.
Fig.9.VariationofSaO2andHbTwithmicrobubblewash-inandwash-outrates.SignificantcorrelationswereobservedforSaO2andHbTwithwash-inrate.Thecorrelations
ofSaO2andHbTwithwash-outratewerenotsignificantatthe0.05level.Thepvaluesindicatetheprobabilitiesthatthecorrelationcoefficients(r)arezero.Symbols
apairofimageproperties(overallROIsandalltumours)andthat for all ROIs for a tumour was obtained, and used to obtain a correctionfactorthatwassubtractedfromeachdependentimage propertyvalueinapair,foreachindividualtumour.Theresulting changes in correlation coefficients and their significance, as indicatedinbrackets(red)inFig.10,suggestthatinter-tumoural variationwasimportantinsomerelationshipsandnotothers.It would therefore be worthwhile continuing such studies with increasedstatistical poweras described in the following para-graph.
Alimitationofthepresentstudywasthattheregistrationofthe MSOTandUSimageswasdependentontheauthor’sjudgementof thematchingfeaturesinthetwosetsofimages,andoffindingan MSOT image that matched the DCE-US imaging plane. Such registrationmayvaryfromusertouser.Importantlyforthepresent study,itslimitedaccuracypreventedapixelbypixelcorrelation betweentheoptoacousticandDCE-USimagepropertiesofthetype achieved by Zion et al. [33], which was possible in their case becausetheyusedhardwareinwhichoptoacousticandultrasound imagingwereimplementedwiththesametransducer.Thislimited thestatisticalpowerof ourintra-tumouralanalyses since large ROIshadtobeused,restrictingthenumberofROIspertumourto betweenfourandseven.Repeatingthisstudyusingsimultaneous optoacoustic and DCE-US imaging with the same transducer should significantly reduce this source of variation, allowing greatly improved statistical power when assessing the intra-tumoural relationships between image characteristics. It also provides motivation for investigating the use of automated registrationmethods,implementingDCE-USontheMSOTsystem andthedevelopmentof3DDCE-US.Thestudyisalsolimitedbythe absenceof histologicalconfirmationof regions of the tumours lackingabloodsignatureonMSOT.Fromthisstudy,noconclusions canbedrawnregardingthehistologicalnatureofsuchregions,e.g. theconditionoftheendothelium,orwhetherthecellsthereare hypoxic,necroticorapoptotic.Infact,thereisageneralneedfor studiesthatusehistologicalmarkersasareferencestandardfor assessingbloodandoxygensaturationusingoptoacousticimages; workinthisareahasjustbegun[50].However,theabsenceofsuch confirmationinthepresentstudydoesnotdetractfromourfinding that in somedarkregions, as imagedby MSOT, thetissue was perfused.
Finally, forpreclinicalcombinedmodalityresearchusingthe iTheraMSOTinVisionTM,thenovelregistrationsystemdescribed maybeofmoregeneralinterest,forregisteringMSOTimagesto variousultrasoundimagessuchasB-mode,microbubble,Doppler and elastography, as well as for registering to high intensity ultrasound and othertherapeutic devices. Additional uses may include the optoacoustic localisation (e.g. for targeting with physicaltherapies)oforthotopictumoursgrownfromcelllines thathavebeengeneticallymodifiedtoexpressproteinsthatare spectroscopicallyidentifiablebyMSOT.
5.Conclusion
Thereisvalueincombiningoptoacousticspectralimagingwith dynamic contrast ultrasound imaging when studying tumour hypoxia. For example, combining the two imaging modalities allowsDCE-UStoassistinterpretationoftheoptoacousticimages, as demonstrated here where DCE-US helped to answer the question of whethera regionthat lacks an optoacousticblood signalisperfused.Eventually,datafromavascularhaemodynamic propertymeasurementmethodsuchasmicrobubbleultrasound may be combined with blood oxygenation and haemoglobin concentration measures from a method such as optoacoustic imaging, in a model-based estimation of the hypoxicstatus of cancercellsinspecificregionsofatumour[23].
IffurtherworkconfirmsthatDCE-USpropertiesarepredictive of low blood oxygen saturation, this finding would suggest potential touseDCE-US characteristics as surrogatesfor blood oxygen saturation, in predicting radiation and drug treatment effectivenessinclinicalsituationswhentumoursarelocatedtoo deepforoptoacousticimagingtobeused.
Conflictofinterest
None.
Acknowledgements
ThisresearchwassupportedbytheEPSRC,theCancerResearch UKCancerImagingCentreattheInstituteofCancerResearchand RoyalMarsdenNHSFoundationTrust,andtheResearchCouncilof
Fig.10.SummaryoftheobservedcorrelationsofoptoacousticimagingpropertiesSaO2(A)andHbT(B)withDCE-UScharacteristicsforallROIsinalltumours.Asterisks
Norway.Weacknowledgefunding(SE,GB)fromCancerResearch UK[grantnumberC309/A11566],EPSRCstrategicequipmentgrant EP/NO15266/1andNHSfundingtotheNIHRBiomedicalResearch Centre. We are grateful to Phoenix Solutions AS, for kindly supplyingtheSonozoidTM.
AppendixA.Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.pacs.2017.08.001.
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AnantShahreceivedhisPh.D.degreeinbiophysicsin 2014fromtheInstituteOfCancerResearch,forhiswork onphotoacousticimagingofmolecularmarkersofcancer prognosisandresponseusinggoldnanoparticles.Heis currently workingasa postdoctoralresearcheratthe Institute ofCancer Research.Hisresearch is oriented towardsassessingthepotentialofphotoacousticimaging forcancertreatmentplanningandtreatmentresponse.
NigelBushisamemberoftheUltrasoundandOptics teamintheDivisionofRadiotherapyandImagingatThe InstituteofCancerResearch(Sutton,UK).Since1979he hasbeeninvolvedinabroadrangeofultrasoundimaging research projects applied to clinical and preclinical studies.Researchfieldsincludeultrasoundskinimaging, elastography,tissuepropertycharacterisation,high fre-quencyarraydevelopmentandUScontrastagents.Heis currentlyusingultrasoundandphotoacousticimagingto monitornovelcontrastagent-drugtherapiestotreat pre-clinicaltumourxenografts.
GaryBoxhasworkedattheInstituteofCancerResearch foroverthirtyyearsandisnowcurrentlyworkingwithin theCancerResearchUKCancerTherapeuticsUnitatthe ICR.Hehasmanyyearsofexperienceindevelopingand optimizing various invitromodels (e.g.invasion and migration assays); evaluating novel compounds for potentialefficacystudies;exvivocultureandgenetically tagging cell lines for bioluminescence imaging and
fluorescenceimaging.
SueEccleswasuntilherrecentretirementProfessorof Experimental Cancer Therapeutics within the CRUK Cancer Therapeutics Unit, The Institute of Cancer Research and now holds the position of Honorary ProfessorofTumourBiologyintheCancerTherapeutics Division.Shewasresponsiblefordirectingthepreclinical evaluation of compounds emerging from the CTU’s molecularly targeted drug discovery programme. Re-search interests include the cellular and molecular mechanismsofmetastasis,focussingon3Dinvitroassays andinvivotumourmodelsystemsandtheroleofthe microenvironment(includinghypoxia)intumour pro-gressionandresistancetotherapy.
