Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies

Sturm, Michael Hess, Stritzker Jochen, Härtl Barbara, Gentschev Ivaylo, Szalay

Description

Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) renal excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells.

Sujets

• Beta-glucuronidase
• Oncolytic virus
• Cancer
• Reporter

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Bacterial

glucuronidase

as

general

marker

for

oncolyticvirotherapyorotherbiologicaltherapies

Hess
etal
.

Hess
etal
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JournalofTranslationalMedicine
2011,
9
:172
http://www.translational-medicine.com/content/9/1/172(11October2011)

Hess
etal
.
JournalofTranslationalMedicine
2011,
9
:172
http://www.translational-medicine.com/content/9/1/172

RESEARCH

OpenAccess

Bacterialglucuronidaseasgeneralmarkerfor
oncolyticvirotherapyorotherbiologicaltherapies
MichaelHess
1
†
,JochenStritzker
1,2,3
†
,BarbaraHärtl
1,2
,JuliaBSturm
1
,IvayloGentschev
1,3
andAladarASzalay
1,3,4*

Abstract
Background:
Oncolyticviraltumortherapyisanemergingfieldinthefightagainstcancerwithrisingnumbersof
clinicaltrialsandthefirstclinicallyapprovedproduct(AdenovirusforthetreatmentofHeadandNeckCancerin
China)inthisfield.Yet,untilrecentlynogeneral(bio)markerorreportergenewasdescribedthatcouldbeusedto
evaluatesuccessfultumorcolonizationand/ortransgeneexpressioninotherbiologicaltherapies.
Methods:
Here,abacterialglucuronidase(GusA)encodedbybiologicaltherapeutics(e.g.oncolyticviruses)was
usedasreportersystem.
Results:
Usingfluorogenicprobesthatwerespecificallyactivatedbyglucuronidasewecouldshow1)preferential
activationintumors,2)renalexcretionoftheactivatedfluorescentcompoundsand3)reproducibledetectionof
GusAintheserumofoncolyticvacciniavirustreated,tumorbearingmiceinseveraltumormodels.Timecourse
studiesrevealedthatreliabledifferentiationbetweentumorbearingandhealthymicecanbedoneasearlyas9
dayspostinjectionofthevirus.Regardingthesensitivityofthenewlydevelopedassaysystem,wecouldshow
thatasingleinfectedtumorcellcouldbereliablydetectedinthisassay.
Conclusion:
GusAthereforehasthepotentialtobeusedasageneralmarkerinthepreclinicalandclinical
evaluationof(novel)biologicaltherapiesaswellasbeingusefulforthedetectionofrarecellssuchascirculating
tumorcells.
Keywords:
beta-glucuronidase,oncolyticvirus,cancer,reporter,fluorescentprobe

Background
DNAintegrationintothehostgenome.Moreover,vac-
Theregainedinterestinoncolyticvirusesoverthepastciniavirusdisplaysabroadhostcellrange,rapidspread
severalyearsledtoanenormousleapinthefieldwithandahighcapacity(upto25kbp)forgeneticpayload
moreandmoreoncolyticvirusestobedescribedandofforeignDNA[3].Ofnoteandimportanceregarding
yettocome.Notonlywerethosevirusesgeneticallythesafetyofvacciniavirus,isalsoitsbillion-foldusein
alteredtoattenuatetheirvirulence,toimprovetheirhumansduringtheeradicationprogramofsmallpox,as
safetyprofileandenhancetheirtumorspecificity,butwellasthefactthatvacciniavirusisnotahuman
theyalsowereequippedwithadditionalgenesfore.g.pathogen.Ontopofthat,recombinantvacciniavirus
cytotoxins,cytokines,prodrugconvertingenzymesandstrains(rVACVs)specificallycolonizesolidtumorsin
reportergenesthatimprovedtheoverallperformanceofmicewhilenotinfectingotherorgans[4-7].Therefore,
theseviruses[1,2].Amongthose,vacciniavirusisoneofitsuseinhumanpatientswaspursuedandfirsthuman
themostpromisingcandidatesandhasseveraladvan-trialshavealreadybeencarriedoutsuccessfully[8-11].
tages:SincethislargeDNAvirusencodese.g.itsownAreliablemonitoringofsuccessfultumorcolonization
DNApolymeraseitisabletoreplicateinthecytoplasminhumanswouldhaveanenormousimpactnotonlyon
ofinfectedhostcellstherebyminimizingtheriskofclinicaltrials,butalsotopredictpossibleoutcomesof
oncolyticvirustherapy.Inthisaspect,weandothers
*Correspondence:[e-mail]
useddifferentkindofreportergenesforoptical(e.g.
1
†
DCeopnarttribmuetnetdoefqBuioalclyhemistry,Biocenter,UniversityofWürzburg,Würzburg,
[5]),orradiological(e.g.[12-15])imagingmodalities.
Germany
Thisenabledvisualizationofvirusreplicationwithinlive
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Hessetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AatntyribmuetidoinumLi,cpernosveid(hetdtpt:h//ecroeriagtiivneaclowmormkoisns.porrogp/leircleyncsitees/db.y/2.0),whichpermitsunrestricteduse,distribution,andreproductionin

Hess
etal
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JournalofTranslationalMedicine
2011,
9
:172
http://www.translational-medicine.com/content/9/1/172

animalmodels.However,asopticalimaginghasstrong
limitationsinpenetrationdepthandradiologicalima-
gingistimeconsumingandrequirestheneedofspecia-
lizedpersonnelandexpensiveequipment,acheapand
simplemethodwithshortturn-around-timesisurgently
needed.Inparticular,ifthismethodcouldalsobeused
inotherbiologicaltherapyapproaches.
Beta-glucuronidasescatalyzethehydrolysisofß-D-
glucuronidesintothecorrespondingD-glucuronateand
alcohol.WhilethemammalianenzymeswithapH-opti-
mumunderacidicconditions(pH4to5)havestrongly
reducedcapacityatnormal(neutral)tissuepH,the
E.
coli
enzymeencodedby
gusA
worksoptimalinthe
rangeofpH6.8to7.7[16].
Itsfirstuseasa(fusion-)reportergenewasdescribedby
Jeffersonetal.[17,18]andwasextensivelyusedinplant
physiologystudies.Inmammals,bacterialglucuronidase
wasmainlyusedinprodrugstudies,duetotheverylow
abundanceofhumanglucuronidaseinhumanserum[19].
Severalstrategiesweresuccessfullyemployed:e.g.fusion
ofcancerspecificantibody-fragmentswithbeta-glucuroni-
dase[20]ortumorselectiveexpressionoftheenzyme
usingbacteria[21]oradenoviruses[22,23].Thereporter
genepropertiesoftheenzymewerenotstudiedasexten-
sivelyinanimals.However,twoindependentapproaches
werepublishedthatlookedatthepotentialofusingbeta-
glucuronidaseasatargetstructureforradiotracersinposi-
tron-emission-tomography[24,25].Inanotherstudy,a
membrane-anchoredformofamouse-glucuronidasewas
usedincombinationwiththefluoresceindi-beta-D-glu-
curonide(FDGlcU)whichwashydrolyzedtoafluorescent
reporterthatcouldbeusedtoassessthelocationandper-
sistenceofgeneexpressioninvivo[26].
Here,weshowthatbeta-glucuronidaseincombination
withfluorogenicsubstratescannotonlybeusedfor
localizationofenzymeexpression,butalsoasageneral
biomarkerforforeignproteinexpressioninserumsam-
ples.Consequently,thedescribedtest-systemcouldbe
appliedtoallkindsofbiologicaltherapieswhichdepend
onheterologousgeneexpression.
Materialsandmethods
Forevaluationofthedescribedglucuronidaseassayitwas
necessarytoconfirmtheheterologousgeneexpressionof
thedescribedvacciniavirusstrainbyWesternblotanalysis
aswellasimmuno-stainingstudiesincellcultureand
infectedtumorsections.Theassayitself(describedinthe
“
Fluorogenicprobesanddetectionoffluorescencepro-
ducts
”
section)wastestedwithpurifiedenzymeaswellas
withsamplesfromvacciniavirusinjectedanimals.
Cellculture
HumanA549lungcancercells(ATCCNo.CCL-185)
wereculturedinRPMI-1640mediumcontaining10%

Page2of11

fetalbovineserum(FBS)and1%antibiotic-antimycotic
solution(PAALaboratories,Cölbe,Germany)under
standardcellcultureconditions(37°C,5%CO
2
).
MTH52cisderivedfromamalignantsmall-cellcanine
carcinomaofthemammarygland[27]andculturedin
DMEMsupplementedwithantibiotic-antimycoticsolu-
tionand20%FBS.
Vacciniaviruses
TheattenuatedvacciniavirusstrainGLV-1h68waspur-
ifiedaspreviouslydescribed[4].Forgenerationofcon-
trolviruses,
lacZ
and
gusA
ofGLV-1h68werereplaced
bynonrelevantgeneconstructstocreatevirusesnega-
tiveforbeta-galactosidase(rVACV-LacZ
-
)andbeta-glu-
curonidase(rVACV-GusA
-
)respectively(AdditionalFile
1,FigureS1).
Infectionofcellcultures
Twodaysbeforeinfection,cellswereseededin6-well
platesforwesternblotanalysisor12-wellplatescon-
tainingsterilecoverslipsformicroscopystudies.90%
confluentcelllayerswereeithermocktreatedor
infectedwithGLV-1h68,rVACV-LacZ
-
orrVACV-
-GusAatamultiplicityofinfection(MOI)of0.1for1h
at37°Cand5%CO
2
inmediumcontaining2%FBS.
Afterwardstheinfectionmediumwasaspiratedand
replacedbystandardcellculturemedium.
Westernblotanalysis
Fordetectionofproteins,infectedcellswereharvested
andlysedinSDSsamplebufferat6,12,24and48
hourspost-infection(hpi).Lysateswereseparatedby
10%SDS-Polyacrylamidegelelectrophoresisandsubse-
quentlytransferredontoanitrocellulosemembrane
(WhatmanGmbH,Dassel,Germany).Afterblockingin
5%skimmilkinPBS,themembranewasincubatedwith
anti-beta-glucuronidaserabbitpolyclonalantibody
(G5420,Sigma-Aldrich,Schnelldorf,Germany),anti-
beta-galactosidaserabbitpolyclonalantibody(A-11132,
MolecularProbes,Leiden,Netherlands),anti-GFPrabbit
polyclonalantibody(sc-8334,SantaCruz,Heidelberg,
Germany)oranti-beta-actinmousemonoclonalanti-
body(ab6276,Abcam,Cambridge,UK).Thefirstanti-
bodiesweredetectedusinghorseradishperoxidase-
conjugatedanti-mouse(ab6728,Abcam,Cambridge,
UK)oranti-rabbit(ab6721,Abcam,Cambridge,UK)
secondaryantibodies,followedbyenhancedchemilumi-
nescencedetection.
X-Gal/X-GlcUstainingandmicroscopystudies
Fortheanalysisofexpressionandactivityofbeta-galac-
tosidaseandbeta-glucuronidaserespectively,A549cells
wereseededoncoverslipsandinfectedwith200PFU
GLV-1h68,rVACV-LacZ
-
orrVACV-GusA
-
perwell.

Hess
etal
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JournalofTranslationalMedicine
2011,
9
:172
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