Hyaluronan-induced masking of ErbB2 and CD44-enhanced trastuzumab internalisation in trastuzumab resistant breast cancer

Hyalu onan-induced masking o E bB2 and CD44-enhanced as uzumab in e naliza ion in as uzumab esis an b eas cance Zsuzsanna Pályi-K ekk1, Má k Ba ok1, Jo ma Isola4, Ma kku Tammi3, János Szöllősi1,2, Pe e Nagy1* 1Depa men o Biophysics and Cell Biology, 2Cell Biophysics Resea ch G oup o he Hunga ian Academy o Sciences, Uni e si y o Deb ecen, 1 Egye em sq , H-4010 Deb ecen, Hunga y 3Depa men o Ana omy, Uni e si y o Kuopio, 70211 Kuopio, Finland 4Ins i u e o Medical Technology, Uni e si y and Uni e si y Hospi al o Tampe e, 33520 Tampe e, Finland Running i le: Masking o E bB2 by hyalu onan Keywo ds: E bB2, as uzumab esis ance, CD44, hyalu onan, masking *Co esponding au ho : Depa men o Biophysics and Cell Biology, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, 1 Egye em squa e, H-4010 Deb ecen, Hunga y. Tel.: +36-52-412623, ax: +36-52-532201, email: [email p o ec ed] 2 Abs ac Al hough as uzumab, a ecombinan humanized an i-E bB2 an ibody, is widely used in he ea men o b eas cance , nei he i s mechanism o ac ion, no he ac o s leading o esis ance a e ully unde s ood. We ha e p e iously shown ha an ibody-dependen cellula cy o oxici y is pi o al in he in i o e ec o as uzumab agains JIMT-1, a cell line showing in i o esis ance o he an ibody, and sugges ed ha masking o he as uzumab-binding epi ope by MUC-4, a cell su ace mucin, ook place. He e, we u he explo ed he ole o masking o E bB2 in connec ion wi h CD44 exp ession and syn hesis o i s ligand, hyalu onan. We show ha high exp ession o CD44 obse ed in JIMT-1 cells co ela es wi h E bB2 down egula ion in i o, while siRNA-media ed inhibi ion o CD44 exp ession leads o dec eased a e o as uzumab in e naliza ion and low cell p oli e a ion in i o. An inhibi o o hyalu onan syn hesis, 4-me hylumbelli e on (4-MU) signi ican ly educed he hyalu onan le el o JIMT-1 cells bo h in i o and in i o leading o enhanced binding o as uzumab o E bB2 and inc eased E bB2 down- egula ion. Fu he mo e, he inhibi o y e ec o as uzumab on he g ow h o JIMT-1 xenog a s was signi ican ly inc eased by 4-MU ea men . Ou esul s poin o he impo ance o he CD44-hyalu onan pa hway in he escape o umou cells om ecep o -o ien ed he apy. 3 In oduc ion O e exp ession o E bB2 has been unques ionably linked o ad e se p ognosis in b eas cance .1 E bB2 he e odime izes wi h o he membe s o he E bB amily o ecep o y osine kinases (RTK) leading o enhanced ligand binding a ini y, p o ec ion om lysosomal deg ada ion and di e si ied signaling.2 E bB2 is iewed as an non-au onomous, ligand-less, posi i e egula o o E bB signaling,2 bu i s pa icipa ion in non-E bB p o ein-media ed signaling is a ac ing mo e and mo e in e es as well. Among hese, β-in eg ins,3 MUC-44 and CD445 a e p obably he bes known candida es whose oles in cance p og ession a e well documen ed.3,6,7 E bB2 is embedded in o his ne wo k o signaling and accesso y molecules and by i s p omiscuous associa ion p o ile i p omo es cance p og ession.8 CD44 is ecognized as he majo hyalu onan ecep o ha ing se e al, al e na i ely spliced iso o ms a ying in hei physiological unc ion.9 Binding o hyalu onan ac i a es CD44- media ed signal ansduc ion pa hways ia in e ac ions be ween CD44, G b2, Va 2 and E bB2.10 CD44 is in ol ed in he di ec egula ion o E bB211 and mul iple o he RTKs.12 I has been sugges ed ha liga ion o CD44 by endogenous hyalu onan leads o he ac i a ion o he phospha idylinosi ol 3-kinase (PI3K)-Ak su i al pa hway,13 and ha displacemen o endogenous hyalu onan by exogenous hyalu onan oligosaccha ides dis up s he ac i a ion.12 CD44-media ed cy oskele al ea angemen s ha e been obse ed6 implying he in ol emen o CD44 in cellula adhesion, mig a ion and in asion.14 CD44 is almos absen in no mal human b eas epi helial cells, eme ges in benign and p emalignan lesions, and is up egula ed in ca cinomas.15 Howe e , a ecen s udy sugges ed ha CD44 opposes a he han p omo es he sp eading o b eas ca cinoma in mice.16 While he ole o CD44 in b eas cance p og ession in i o is no comple ely se led, he accumula ion o hyalu onan a ound malignan cells o in adjacen s oma has been unambiguously shown o be an indica o o poo p ognosis in b eas cance .17 In line wi h he indings on human umou s, hyalu onan syn hesis acili a es he in asi e g ow h o g a ed umou cells in i o,18 and blocking hyalu onan in e ac ions wi h i s ecep o s, using soluble CD44 o hyalu onan oligome s, inhibi s umou cell g ow h in expe imen al animals.19 Besides c ea ing signals o p e en apop osis 13, hyalu onan is equi ed o ac i a ion o he E bB2-E bB3 ecep o leading o he o ma ion o ca diac al es.20 Thus, he e is moun ing e idence ha E bB2, CD44 and hyalu onan a e connec ed bo h in physiological signaling as well as cance pa hogenesis h ough mechanisms la gely unknown a p esen .5,11 4 Being a memb ane p o ein and a key membe o su i al and p oli e a ion signaling pa hways E bB2 is he a ge o ecep o -o ien ed an ibody he apy.21 T as uzumab (He cep in), a humanized monoclonal an i-E bB2 an ibody, induces objec i e clinical esponses in 40% o pa ien s as a single agen gi en as i s -line ea men o E bB2- o e exp essing me as a ic b eas cance .22 Al hough combina ion o as uzumab wi h con en ional chemo he apy inc eases esponse a es d ama ically, he de elopmen o esis ance seems cu en ly ine i able.23 Di ec ac ion o as uzumab on E bB2 (e.g. E bB2 down- egula ion, inac i a ion o Ak , inhibi ion o me allop o ease-media ed shedding)24 and an ibody-dependen cellula cy o oxici y (ADCC)25,26 ha e been in oked o explain he mechanism o ac ion o as uzumab. Despi e in ense in es iga ions as uzumab esis ance emains enigma ic and unp edic able in he clinical se ing. P oduc ion o EGF-like g ow h ac o s,27 loss o PTEN,28 masking o E bB24 and impai ed ADCC eac ion29 ha e all been sugges ed as possible mechanisms. In he cu en pape we in es iga ed JIMT-1, a cell line showing in i o esis ance o as uzumab,30 and ound a high le el o CD44 o e exp ession. We showed p e iously ha he in i o as uzumab esis ance o he cell line is pa ial, and de elopmen o comple e as uzumab esis ance akes 5-10 weeks.26 We sugges ed ha masking o E bB2 may be he culp i .4 Gi en he sugges ed associa ion o CD44 wi h E bB211 we asked whe he CD44 plays any signi ican ole in he su i al o JIMT-1 du ing as uzumab he apy. Using siRNA-media ed supp ession o CD44 exp ession we showed ha CD44 is necessa y o as uzumab-induced in e naliza ion o E bB2 and o he su i al o JIMT-1 cells in i o. 4- me hylumbelli e one (4-MU), a hyalu onan syn hase inhibi o , has been shown o inc ease he e iciency o chemo he apy.31 We easoned ha hyalu onan may play a ole in masking o cell su ace E bB2.32 We show ha in i o and in i o ea men wi h 4-MU dec eased he pe icellula hyalu onan concen a ion in JIMT-1 xenog a s accompanied by inc eased binding o as uzumab o E bB2. 4-MU ac ed syne gis ically wi h as uzumab in inhibi ing he p og ession o JIMT-1 umou s. Elucida ion o he ole o CD44 o e exp ession in as uzumab esis an cell lines may help unde s and he causes o he apeu ic ailu es in pa ien s wi h his ype o b eas cance . 5 Ma e ials and Me hods Cells. JIMT-1 cells we e g own in F-12/ DMEM (1:1) supplemen ed wi h 10% FCS, 60 uni s/L insulin and an ibio ics.30 The SKBR-3 cell line was ob ained om he Ame ican Type Cul u e Collec ion (Rock ille, MD) and g own acco ding o i s speci ica ions. An ibodies. T as uzumab (He cep in) was pu chased om Roche L d. (Budapes , Hunga y). Mab 2C4 was a gene ous gi om Genen ech (Sou h San F ancisco, CA). Monoclonal an ibodies agains E bB2 (E bB2-76.5) and CD44 (He mes-3) we e p oduced om hei hyb idoma supe na an s (E bB2-76.5 ob ained om Y. Ya den, Weizmann Ins i u e o Science, Reho o , Is ael; He mes-3 p oduced by he HB-9480 hyb odima ob ained om ATCC) and pu i ied using p o ein A a ini y ch oma og aphy. He mes-3 was kindly dona ed by D . Si pa Jalkanen (Uni e si y o Tu ku, Finland). Cy3- and Cy5-conjuga ed goa an i human IgG (H+L) Fab was ob ained om Jackson ImmunoResea ch Eu ope (Camb idgeshi e, UK). Conjuga ion o p ima y an ibodies wi h AlexaFluo (Molecula P obes, Eugene, OR), Cy3 and Cy5 (Ame sham, B aunschweig, Ge many) dyes was ca ied ou acco ding o he manu ac u e s’ speci ica ions. Hyalu onan. Highly pu i ied la ge molecula weigh hyalu onan (HA-LMW) wi h an a e age molecula mass o 1.2×106 Da was dona ed by Genzyme (Camb idge, MA). Pu i ied hyalu onan decasaccha ides (HA10) we e kindly p o ided by Seikagaku Co po a ion (Tokio, Japan). Wes e n blo ing. Whole cell lysa es we e p epa ed in lysis bu e con aining 20 mM T is- HCl, pH 7.5, 150 mM NaCl, 10% glyce ol, 1 mM EGTA, 1 % T i on X-100, 1 Comple e Mini (Roche, Mannheim, Ge many) p o ease inhibi o cock ail able /10 mL, 1 mM Na3VO4, 1 mM PMSF, 10 mM NaF, 10 mM β-glyce ol phospha e and 10 mM Na4P2O7. Immunop ecipi a ion o E bB2 and E bB1 we e ca ied ou wi h Ab3-OP15 an ibody (Calbiochem-Me ck Biosciences, Schwalbach, Ge many) and F4 (E3138, Sigma, Schnelldo , Ge many), espec i ely, o 1 h on ice and Sepha ose 4B Fas Flow P o ein G beads (Sigma). Immunop ecipi a es we e esol ed on SDS-polyac ylamide gels and blo ed o ni ocellulose memb anes. The ollowing an ibodies we e used o p ima y labelling o he memb anes a dilu ions sugges ed by he manu ac u e s: Ab3-OP15 o E bB2, F4 o E bB1, PY99-sc7020 (San a C uz Bio echnology, San a C uz, CA) o phospho y osine and He mes- 6 3 o CD44. Pe oxidase-conjuga ed goa an i-mouse IgG and an enhanced chemiluminescence ki (Ame sham, F eibu g, Ge many) we e used o de ec ion. Flow cy ome ic measu emen o cell numbe s and ecep o exp ession le els. Quan i a i e de e mina ion o ecep o exp ession le els was ca ied ou on a FACSCalibu low cy ome e (Bec on Dickinson, F anklin Lakes, NJ) using Qi iki (DakoCy oma ion, Glos up, Denma k) acco ding o he manu ac u e ’s ins uc ions. In p oli e a ion assays cells we e coun ed wi h a FacsA ay low cy ome e (Bec on Dickinson). Xenog a umou s. The se e e combined immunode iciency (SCID) C.B-17 scid/scid mouse popula ion o igina ed om he labo a o y o Fox Chase Cance Cen e , Philadelphia, PA, and we e housed in a pa hogen- ee en i onmen . Only nonleaky mice wi h mu ine IgG le els below 100 ng/ml we e used in his s udy. Se en-week old emale SCID mice we e gi en a single subcu aneous injec ion o 5×106 JIMT-1 cells suspended in 150 μl Hank’s bu e and mixed wi h an equal olume o Ma igel (Basemen Memb ane Ma igel, BD Biosciences, Bed o d, MA). Tumou olumes we e calcula ed as he p oduc o he leng h, wid h and heigh o he umou measu ed once a week wi h a calipe . T as uzumab was adminis e ed a a dose o 5 μg/g by weekly in ape i oneal (i.p.) injec ion. Con ol mice ecei ed weekly i.p. injec ion o 100 µl physiologic saline. Animals we e eu hanized by CO2 inhala ion. The expe imen s we e done wi h he app o al o he e hical commi ee o he Uni e si y o Deb ecen. 4-me hylumbelli e one ea men . 4-me hylumbelli e one (4-MU) (Sigma, Budapes , Hunga y), an inhibi o o hyalu onan syn hase, was suspended in 1% a abic gum and adminis e ed o ally a a dose o 3 mg/g body weigh wice daily.33 Fo in i o expe imen s 4- MU was dissol ed in PBS and added o he cul u e medium a a concen a ion o 1 mM. Immunohis ochemis y. Subcu aneous umou s we e emo ed om anes he ized mice, co e ed wi h Shandon C yoma ix (The mo Elec on Co po a ion, Wal ham, MA) and s o ed in liquid ni ogen. 20-µm hick as ozen samples we e made by Shandon AS-620E C yo ome (The mo Elec on Co po a ion) on silanized slides, ixed in 4% o maldehyde o 30 min, and washed wice in PBS (pH 7.4) supplemen ed wi h 1% BSA o 20 min a oom empe a u e. The slides we e labeled wi h a sa u a ing concen a ion (10-20 µg/ml) o luo opho e-conjuga ed an ibodies in 100 µl PBS con aining 1% BSA (PBS-BSA) o e nigh on ice. The samples we e washed wice wi h PBS-BSA and co e ed wi h 15 µl Mowiol (Me ck, Budapes , Hunga y). Fo labelling o hyalu onan issue sec ions we e ea ed wi h endogenous bio in blocking ki (Molecula P obes) ollowed by labelling wi h 5 μg/ml 7 bio inyla ed HABC (hyalu onan binding complex) a 4 oC o e nigh .34 P io o s aining wi h luo escein-a idin he samples we e washed i e imes in PBS-BSA. Con ocal mic oscopy. A Zeiss LSM 510 con ocal lase -scanning mic oscope (Ca l Zeiss AG, Gö ingen, Ge many) was used o image samples. AlexaFluo 488 was exci ed a 488 nm and de ec ed be ween 505-530 nm. Cy3 and AlexaFluo 546 we e exci ed wi h he 543 nm line o a g een He-Ne lase , and emission was measu ed be ween 560-615 nm. Cy5 and AlexaFluo 647 we e exci ed wi h he 633 nm line o a ed He-Ne lase , and hei emissions we e measu ed o e 650 nm. Fluo escence images we e aken as 1-μm op ical sec ions using a 63x (NA=1.4) oil imme sion objec i e. Image analysis. Con ocal mic oscopic images we e analyzed wi h he DipImage oolbox (Del Uni e si y o Technology, Del , The Ne he lands) unde Ma lab (Ma hwo ks Inc., Na ick, MA). The cell memb ane was iden i ied by a manually-seeded wa e shed algo i hm35 using a cus om- w i en in e ac i e algo i hm implemen ed in Dipimage/Ma lab. The luo escence in ensi y was e alua ed only in pixels co esponding o he cell memb ane. Fo he analysis o he in luence o CD44 exp ession on he ela i e binding o as uzumab o E bB2 issue sec ions we e iple- labeled wi h AlexaFluo 488-E bB2-76.5, Cy3-an i-human IgG ( o isualize as uzumab) and AlexaFluo 647-He mes-3 (agains CD44). A wo-dimensional his og am (do plo ) o as uzumab s. E bB2 in ensi y was p epa ed using only memb ane pixels. The CD44 exp ession le el was sepa a ely e alua ed in pixels co esponding o high as uzumab/E bB2 and low as uzumab/E bB2 a ios iden i ied based on he do plo . Colocaliza ion be ween wo di e en luo escen labels was calcula ed acco ding o Pea son’s o mula: ()( ) ()( ) ∑∑ ∑ −− −− kmeank kmeank kmeankmeank JJII JJII 22 whe e Ik and Jk a e he in ensi ies o he k h pixel in he i s and second image, espec i ely, Imean and Jmean a e he a e age luo escence in ensi ies o he i s and second image, espec i ely. Low in ensi y pixels we e excluded om he analysis. The alue o he c oss- co ela ion coe icien anges om +1 o −1. Values close o 1, 0 and −1 indica e high and low deg ee o colocaliza ion, and an ico ela ion, espec i ely. Colocaliza ion was e alua ed using a cus om-made so wa e w i en in LabView (Na ional Ins umen s, Aus in, TX). Fluo escence esonance ene gy ans e (FRET). Flow cy ome ic FRET measu emen s we e pe o med on a FACSVan age SE ins umen wi h DiVa op ion (Bec on Dickinson) 8 equipped wi h h ee lase s emi ing a 488, 532 and 633 nm. A de ailed desc ip ion o he me hod has been published elsewhe e.36 Cellula au o luo escence was measu ed in he FL1 channel (exci ed by he 488 nm lase ) h ough a 530/30 nm band pass il e o disca d deb is and dead cells. Dono luo escence was exci ed a 532 nm and eco ded in he FL4 channel h ough a 585/42 nm bandpass il e , while he accep o was exci ed a 633 nm and de ec ed in he FL6 channel h ough a 650 nm long pass il e . The FRET in ensi y was exci ed a 532 nm and de ec ed in he FL5 channel h ough a 650 nm long pass il e . Calcula ions we e ca ied ou on a cell-by-cell basis using he ReFlex so wa e.37 FRET e iciencies we e no malized o a 1:1 dono -accep o a io38 and a e p esen ed as mean alues o FRET his og ams o 10,000 cells. In e naliza ion o as uzumab. siRNA- ans ec ed and con ol cells we e incuba ed wi h 20 µg/ml AlexaFluo 647-labeled as uzumab a 37°C. The samples we e ea ed wi h acid s ip bu e (0.5 M NaCl, 0.1 M glycine, pH 2.5) o 3 minu es on ice ollowed by washing and esuspension in PBS. Cells we e analyzed by low cy ome y, and he in e nalized ac ion o as uzumab was calcula ed by di iding he mean luo escence in ensi y o he acid-s ipped sample wi h ha o he non-acid- ea ed con ol. RNA in e e ence (RNAi). Small in e e ing RNA (siRNA) agains human CD44 we e designed and syn hesized by Dha macon (Chicago, IL) using he SMARTselec ion ules. The sequences o he an i-sense s ands o he siRNAs a e: AUGUCUUCAGGAUUCGUUCUU (CD44 siRNA-4), UAUUCAAAUCGAUCUG CGCUU (CD44 siRNA-5). An siRNA agains GFP was used as a nega i e con ol.39 siRNA ans ec ion o JIMT-1 was ca ied ou wi h he Nucleo ec o de ice o Amaxa (Cologne, Ge many) acco ding o he manu ac u e ’s speci ica ions. The op imal elec opo a ion condi ions (solu ion V, p og am T-20) we e selec ed using GFP plasmid ans ec ion. 9 Resul s CD44 is o e exp essed on as uzumab esis an JIMT-1 cells and associa ed wi h E bB2. We compa ed he exp ession le els o CD44 in as uzumab esis an and sensi i e b eas cance cell lines in o de o e eal he possible oles o CD44 in as uzumab esis ance. Flow cy ome ic da a showed a ~35- imes highe exp ession le el o CD44 in as uzumab esis an JIMT-1 cells (2.3±0.3 million/cell) han in SKBR-3 (65000±5000/cell), hei as uzumab-sensi i e coun e pa (Fig.1A). Since CD44 has been shown o in e ac wi h E bB2 in o a ian cance ,5,10 we in es iga ed he po en ial in e ac ion be ween hem. Analysis o con ocal mic oscopic images yielded c oss-co ela ion coe icien s o 0.612 and 0.602 be ween CD44 and E bB2 on JIMT-1 and SKBR-3 cells, espec i ely (Fig.1B,C). The c oss- co ela ion coe icien be ween wo di e en an ibodies agains E bB2 was used as a posi i e con ol. The ac ha he c oss-co ela ion coe icien o he posi i e con ol was no subs an ially di e en om ha be ween CD44 and E bB2 implies a s ong colocaliza ion be ween hese molecules on he mic ome e scale. No malized low cy ome ic luo escence esonance ene gy ans e (FRET) alues o 16±3% and 10±3% o he associa ion o CD44 and E bB2 in JIMT-1 and SKBR-3, espec i ely, show ha hese molecules a e associa ed a he molecula le el as well (Fig.1B), since FRET alues abo e 5% a e conside ed o imply signi ican associa ion.40 The abo e biophysical da a demons a ing associa ion be ween E bB2 and CD44 in JIMT-1 cells we e ein o ced by molecula biological me hods. E bB2 co-immunop ecipi a ed wi h CD44 (Fig. 1D, le panel), and i s y osine phospho yla ion was inc eased by la ge molecula weigh hyalu onan, whe eas hyalu onan decasaccha ide inhibi ed E bB2 y osine phospho yla ion (Fig. 1D, middle panel). Nei he slow, no la ge molecula weigh hyalu onan modi ied he ac i a ion s a e o E bB1 (Fig. 1D, igh panel). CD44 exp ession co ela es wi h as uzumab in e naliza ion in JIMT-1 xenog a s. In o de o s udy he possible ole o CD44 o e exp ession in as uzumab esis ance se en- week old emale SCID mice we e inocula ed by JIMT-1 cells wi h a single subcu aneous injec ion. Mice ecei ed as uzumab o physiologic saline weekly s a ing immedia ely a e umou injec ion. Mice we e ea ed wi h as uzumab o 9 weeks, hen by physiologic saline o ano he 6 weeks be o e sac i icing. Tumou sec ions we e iple-s ained agains E bB2, as uzumab and CD44. The wa e shed algo i hm was used o segmen he images. The seeds o he wa e shed algo i hm we e placed inside cells posi i e o CD44 and E bB2. Since mouse s omal cells exp ess nei he CD44 no E bB2, his app oach ensu ed ha he analysis was es ic ed o JIMT-1 cells. We obse ed a lack o igh co ela ion be ween as uzumab binding and E bB2 exp ession in hese mice whose as uzumab he apy had 16 Acknowledgemen s We a e indeb ed o D . Is án Juhász o allowing access o he animal ca e acili y o he Depa men o De ma ology, Uni e si y o Deb ecen, o D . Si pa Jalkanen (Uni e si y o Tu ku, Finland) o p o iding he He mes-3 an ibody and o Gábo Ho á h o helping in he FRET measu emen s. We g a e ully acknowledge he excellen echnical assis ance o Fe enc Bos yán in he animal expe imen s. G an suppo : Hunga ian Academy o Sciences (OTKA F049025, T043061, K62648) and Eu opean Commission (LSHB-CT-2004-503467, LSHC-CT-2005-018914) g an s o PN, JS; Academy o Finland # 107173 (MT) Con lic o in e es None decla ed. 17 Re e ences 1. 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J In es De ma ol 2004;123:708-714. 21 Figu e legends: Figu e 1 CD44 is o e exp essed in JIMT-1 cells and in e ac s wi h E bB2 A. JIMT-1 (g ey dashed line) and SKBR-3 (black dashed line) cells we e labeled wi h AlexaFluo 488-He mes-3 an ibody agains CD44, and he luo escence in ensi y was measu ed by low cy ome y. Unlabeled JIMT-1 (g ey con inuous line) and SKBR-3 (black con inuous line) cells we e used as a nega i e con ol. B. JIMT-1 and SKBR-3 cells we e labeled wi h Cy3-2C4 (agains E bB2) and Cy5-He mes-3 (agains CD44), and he FRET e iciency was measu ed by low cy ome y (black ba s) and he c oss-co ela ion coe icien by con ocal mic oscopy (g ey ba s). Cells labeled wi h wo non-compe ing an ibodies agains E bB2 (Cy3- as uzumab, Cy5-2C4) we e used as a posi i e con ol o bo h FRET and c oss-co ela ion measu emen s. E o ba s indica e he s anda d e o o he mean. C. JIMT-1 cells we e labeled wi h AlexaFluo 488-2C4 and AlexaFluo 647-He mes-3 agains E bB2 and CD44, espec i ely. The ed and g een channels co espond o E bB2 and CD44, espec i ely. The yellow colo ep esen s o e lap be ween he wo luo escence channels. Quan i a i e e alua ion o colocaliza ion is shown in pa B. D. Le panel: JIMT-1 cell lysa e was immunop ecipi a ed wi h He mes-3 (agains CD44), and he memb ane was blo ed wi h He mes-3 and OP15 (agains E bB2). Middle and igh panels: JIMT-1 cells we e s imula ed wi h 100 μg/ml hyalu onan decasaccha ide (HA10) o la ge molecula weigh hyalu onan (HA-LMW) o 30 min a 37 oC. E bB2 (middle panel) and E bB1 ( igh panel) we e immunop ecipi a ed wi h OP15 and F4, espec i ely. The memb anes we e p obed wi h OP15, F4 and PY99 o de ec E bB2, E bB1 and phospho y osine, espec i ely. Figu e 2 CD44 enhances as uzumab in e naliza ion in JIMT-1 xenog a s A-E. Immuno luo escen s aining o JIMT-1 xenog a s samples was ca ied ou six weeks a e inishing as uzumab ea men . Sec ions we e iple-s ained wi h AlexaFluo 488- E bB2-76.5, Cy3-an i-human IgG ( ecognizing as uzumab) and AlexaFluo 647-He mes-3 (agains CD44). The cell memb ane iden i ied by a manually-seeded wa e shed algo i hm is shown in ed (A). Dual colo images show he co ela ion o signals (B: ed – E bB2, g een – as uzumab; C: ed – CD44, g een – as uzumab). Ga e 1 and 2 iden i y egions in he as uzumab-E bB2 con ou plo (D) co esponding o pixels wi h high and low as uzumab- 22 E bB2 a ios, espec i ely. The CD44 in ensi y dis ibu ions co esponding o ga e 1 and 2 a e shown in pa E. F. Mice xenog a ed wi h JIMT-1 umou s we e ea ed by as uzumab o 15 weeks and umou samples we e s ained wi h AlexaFluo 488-E bB2-76.5 and Cy3-an i-human IgG ecognizing as uzumab. The con ou plo shows a much s onge co ela ion be ween he E bB2 and as uzumab signals compa ed o ha obse ed in pa D. G-I. Mice injec ed wi h JIMT-1 cells we e ea ed wi h saline (G) o as uzumab (H) o 15 weeks. In ano he g oup mice we e sac i iced 6 weeks a e a 9-week long as uzumab ea men was s opped (I), and he co ela ion be ween CD44 and E bB2 exp ession le els was analyzed in he h ee samples. Figu e 3 siRNA-media ed supp ession o CD44 exp ession inhibi s as uzumab in e naliza ion A. JIMT-1 cells we e ans ec ed wi h an i ele an siRNA agains GFP (black), CD44 siRNA-4 (whi e) and CD44 siRNA-5 (g ey). The ba s show he exp ession le els o CD44 and an i ele an p o ein, MHC-I, calcula ed om low cy ome ic his og ams analyzed 48 hou s a e ans ec ion. B. In e naliza ion o as uzumab was measu ed in mock- ans ec ed cells (●), cells ans ec ed wi h GFP siRNA (○), CD44 siRNA-4 (▼) o CD44 siRNA-5 (◊). The ac ion o in e nalized as uzumab (100%=amoun o as uzumab bound o he cell su ace a 0 min) is plo ed as a unc ion o he du a ion o as uzumab ea men . The 30 min alues o bo h CD44 siRNA- ans ec ed samples we e s a is ically signi ican ly di e en om he GFP siRNA- ans ec ed one (S uden ’s es , p<0.05). E o ba s indica e he s anda d e o o he mean. Figu e 4 4-me hylumbelli e one ac s syne gis ically wi h as uzumab in inhibi ing umou g ow h and down- egula ing E bB2 exp ession A. Mice we e xenog a ed wi h JIMT-1 cells, and ea ed wi h saline (●), as uzumab (○), 4- me hylumbelli e one (▼) o as uzumab+4-me hylumbelli e one (◊). 4-me hylumbelli e one ea men was ca ied ou daily. A he imes o weekly saline and as uzumab ea men s indica ed by he a ows he size o umou s was measu ed, and i is displayed as unc ion o ime. B. Tissue sec ions we e iple-labeled wi h bio in-HABC and luo escein-a idin o isualize hyalu onan, and an ibodies agains CD44 and E bB2. The cell memb ane o JIMT-1 cells was 23 iden i ied by he manually-seeded wa e shed algo i hm. The mean (±s anda d e o o he mean) exp ession le els o hyalu onan, CD44 and E bB2 measu ed a ound he cell memb ane in as uzumab (whi e), 4-me hylumbelli e one (le -ha ched) and as uzumab+4- me hylumbelli e one (g ey) ea ed animals we e no malized o he saline- ea ed xenog a samples (black). The means we e calcula ed om 6 images aken o issue sec ions o 3 animals. Figu e 5 Inhibi ion o hyalu onan syn hase inc eases binding o as uzumab o E bB2 A-C. JIMT-1 umou samples o saline- ea ed animals we e s ained o hyalu onan wi h bio in-HABC (A) and o CD44 (B). The colo composi e image shows he o e lap o he dis ibu ion o he hyalu onan (g een) and CD44 ( ed) signals. D-F. Tumou sec ions o mice ea ed wi h as uzumab (D) o as uzumab+4- me hylumbelli e one (E) we e s ained wi h an i-human IgG ( ecognizing as uzumab, g een) and E bB2-76.5 ( ed) an ibodies. The con ou plo (F) shows inc eased binding o as uzumab o E bB2 in samples aken om he as uzumab+4-me hylumbelli e one- ea ed animals ( ed con ou s) compa ed o he as uzumab- ea ed ones (black con ou s). The axes o he con ou plo show luo escence in ensi y alues o as uzumab and E bB2-76.5 an ibodies eco ded in memb ane pixels which we e iden i ied by manually-seeded wa e shed segmen a ion. G. Cul u ed JIMT-1 cells we e ea ed wi h 4-me hylumbelli e one o 2 days. Bo h con ol (black con ou s) and 4-me hylumbelli e one- ea ed ( ed con ou s) cells we e s ained wi h AlexaFluo 488- as uzumab and AlexaFluo 647-2C4. The la e an ibody measu ed he amoun o E bB2. Figu e 6 RNA in e e ence-media ed supp ession o CD44 exp ession inhibi s p oli e a ion o JIMT-1 cells An equal numbe o mock- ans ec ed cells (whi e ba s) and cells ans ec ed wi h GFP siRNA (black ba s) o CD44 siRNA-4 (g ey ba s) we e seeded in o 6-well pla es 24 hou s a e ans ec ion, and cul u ed in he absence (– as ) o p esence (+ as ) o 20 μg/ml as uzumab o 72 hou s. Cell numbe s a e no malized o he numbe ound in mock- ans ec ed, as uzumab-un ea ed cells 72 hou s a e ans ec ion. JIMT-1 SKBR-3 pos. con . FRET e iciency (%) 0 5 10 15 20 25 30 C ossco ela ion coe icien 0.0 0.2 0.4 0.6 0.8 1.0 Fig. 1 Pályi-K ekk e al. B Fluo escence in ensi y 1 10 100 1000 10000 Rela i e equency o cells 0.000 0.005 0.010 A JIMT-1, He mes-3 SKBR-3, He mes-3 unlabeled JIMT-1 and SKBR-3 cells E bB2 0 10000 20000 30000 40000 50000 T as uzumab 0 10000 20000 30000 40000 50000 60000 70000 F Fig. 5F-G Pályi-K ekk e al. E bB2 0 1000 2000 3000 T as uzumab 0 1000 2000 3000 G Rela i e numbe o cells (%) 0 20 40 60 80 100 120 140 Fig. 6 Pályi-K ekk e al. JIMT-1 SKBR-3 + as .- as . + as .- as .