In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe

Ci a ion: Melemenidis, S.; Knigh , J.C.; Ke semans, V.; Pe ez-Balde as, F.; Za ghami, N.; So o, M.S.; Co nelissen, B.; Muschel, R.J.; Sibson, N.R. In Vi o PET De ec ion o Lung Mic ome as asis in Mice by Ta ge ing Endo helial VCAM-1 Using a Dual-Con as PET/MRI P obe. In . J. Mol. Sci. 2024, 25, 7160. h ps://doi.o g/10.3390/ ijms25137160 Academic Edi o : Mi-Ae Pa k Recei ed: 28 May 2024 Re ised: 21 June 2024 Accep ed: 26 June 2024 Published: 28 June 2024 Copy igh : © 2024 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). In e na ional Jou nal o Molecula Sciences A icle In Vi o PET De ec ion o Lung Mic ome as asis in Mice by Ta ge ing Endo helial VCAM-1 Using a Dual-Con as PET/MRI P obe S a os Melemenidis 1 , James C. Knigh 2 , Vee le Ke semans 3 , F ancisco Pe ez-Balde as 4 , Nilou a Za ghami 5 , Manuel Sa mien o So o 6, Ba Co nelissen 7, Ru h J. Muschel 5and Nicola R. Sibson 5,* 1Depa men o Radia ion Oncology, S an o d School o Medicine, Cance Ins i u e, S an o d Uni e si y, S an o d, CA 94305, USA; s a mel@s an o d.edu 2School o Na u al and En i onmen al Sciences, Newcas le Uni e si y, Newcas le upon Tyne NE1 7RU, UK; [email p o ec ed] 3Clinical Nuclea Medicine Imaging, Siemens Heal hinee s, 2595 BN The Hague, The Ne he lands; [email p o ec ed] 4Cu eVac, AG. Paul-Eh lich-S . 15, 72076 Tubingen, Ge many; anciscope [email p o ec ed] 5Depa men o Oncology, Uni e si y o Ox o d, Ox o d OX3 7DQ, UK; [email p o ec ed] (N.Z.); u [email protected] (R.J.M.) 6Depa men o Biochemis y and Molecula Biology, Uni e si y o Se ille, 41004 Se ille, Spain; [email p o ec ed] 7Depa men o Nuclea Medicine, Uni e si y Medical Cen e G oningen, Hanzeplein 1, 9713 GZ G oningen, The Ne he lands; ba .co [email protected] *Co espondence: [email protected]; Tel.: +44-1865-225836; Fax: +44-1865-857127 Abs ac : Cu en clinical diagnos ic imaging me hods o lung me as ases a e sensi i e only o la ge umou s (1–2 mm c oss-sec ional diame e ), and ea ly de ec ion can d ama ically imp o e ea men . We ha e p e iously demons a ed ha an an ibody- a ge ed MRI con as agen based on mic opa icles o i on oxide (MPIO; 1 µ m diame e ) enables he imaging o endo helial ascula cell adhesion molecule-1 (VCAM-1). Using a mouse model o lung me as asis, up egula ion o endo helial VCAM-1 exp ession was demons a ed in mic ome as asis-associa ed essels bu no in no mal lung issue, and binding o VCAM-MPIO o hese essels was e iden his ologically. Owing o he lack o p o on MRI signals in he lungs, we modi ied he VCAM-MPIO o include zi conium-89 ( 89 Z , 1/2 = 78.4 h) in o de o allow he in i o de ec ion o lung me as ases by posi on emission omog aphy (PET). Using his new agen ( 89 Z -DFO-VCAM-MPIO), i was possible o de ec he p esence o mic ome as ases wi hin he lung in i o om ca. 140 µ m in diame e . His ological analysis combined wi h au o adiog aphy con i med he speci ic binding o he agen o he VCAM-1 exp essing ascula u e a he si es o pulmona y mic ome as ases. By e aining he o iginal VCAM- MPIO as he basis o his new molecula con as agen , we ha e c ea ed a dual-modali y (PET/MRI) agen o he concu en de ec ion o lung and b ain mic ome as ases. Keywo ds: mic ome as asis; ea ly de ec ion; mic opa icles o i on oxide (MPIO); ascula cell adhesion molecule-1 (VCAM-1); dual modali y (PET/MRI) 1. In oduc ion Me as asis om a p ima y umou si e o dis an o gans is a complex succession o e en s collec i ely known as he in asion-me as asis cascade [ 1 ]. Cu en ly me as asis accoun s o 90% o human cance dea hs and is one o he majo challenges in cance he apy [2] . Owing o he capaci y o cance cells o sp ead o he lungs, his is he sec- ond mos equen si e o me as asis, wi h an es ima ed 20 o 54% o umou s o igina ing elsewhe e in he body me as asising o his o gan [ 3 ]. The mos p e alen cance s ha me as asise o he lung pa enchyma include hose o igina ing om he b eas , lung, colon, In . J. Mol. Sci. 2024,25, 7160. h ps://doi.o g/10.3390/ijms25137160 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2024,25, 7160 2 o 19 u e ine leiomyosa coma, and head and neck squamous cell ca cinomas [ 3 , 4 ]. In child en, i is a e o see p ima y lung cance , and he majo i y o lung cance me as asis cases me as- asise om a p ima y si e o he lungs [ 5 ]. The mos equen ly employed non-in asi e imaging modali ies o iden i ying lung me as ases a e compu ed omog aphy (CT) wi h o wi hou he applica ion o con as agen s and 18 F luo odeoxyglucose ( 18 F-FDG) posi on emission omog aphy (PET)/CT scans. Al hough he imp o emen in CT echnologies allows o spa ial esolu ion de ec ion o lung nodules below 5 mm, i canno p o ide c i e ia o p edic he na u e o hese nodules [ 6 ]. 18 F-FDG PET complemen s he CT es- olu ion, o e ing in o ma ion abou unce ain small lesions and hei me abolic ac i i y. I is impo an o no e, howe e , ha he de ec ion sensi i i y o 18 F-FDG PET elies on he accumula ion o highly me abolically ac i e cells (bo h p oli e a ing umou cells and in lamma o y cells) o allow umou de ec ion/diagnosis, which ypically e lec s a ela- i ely la e s age o umou de elopmen and lea es mic ome as ases unde ec ed [7,8] . This esolu ion limi a ion esul s in dec eased sensi i i y o low- olume diseases, leading o po en ial alse nega i es [ 9 , 10 ]. Mo eo e , CT scans depend on size and densi y con as o iden i y abno mali ies and such con as s may no be e iden in ea ly-s age disease, causing mic ome as ases o be o e looked [ 11 ]. A he same ime, 18 F-FDG is no exclu- si e o umou s and accumula es in any glucose-a id issue, including hose a ec ed by in lamma ion. This non-speci ic up ake can obscu e he p esence o a disease o mimic i , complica ing he di e en ia ion be ween cance ous and non-cance ous condi ions [ 12 , 13 ]. Owing o he lack o imaging sensi i i y o he ea ly s ages o umou cell ex a asa ion om ci cula ion and mic ome as a ic o ma ion, clinical he apy is o en applied a a ime ha is beyond he poin o e ec i e in e en ion. Thus, signi ican ad an ages may be gained i mo e sensi i e imaging me hods can be de eloped o allow molecula a ge ing o ma ke s ha enable he ea ly de ec ion o mic ome as a ic colonies. We ha e p e iously demons a ed ha i is possible o de ec mic ome as ases in mouse b ain e y ea ly in hei de elopmen using molecula ly a ge ed magne ic esonance imaging (MRI) ia he associa ed endo helial ac i a ion and up egula ion o endo ascula adhesion molecules [ 14 – 16 ]. These endo helial adhesion molecules a e apidly up egula ed in esponse o disease o inju y and media e leukocy e olling, adhesion, and ansmig a ion ac oss he ascula wall [ 17 ]. The e is now e idence o sugges ha umou cells also use such adhesion molecules o p omo e hei adhesion o and ex a asa ion ac oss he ascula endo helium [ 18 ]. One such adhesion molecule is ascula cell adhesion molecule 1 (VCAM-1), which is up egula ed e y ea ly in b ain me as asis o ma ion [ 19 ] and has also been shown o enhance endo helial cell ec ui men o new blood essel o ma ion, hus suppo ing umou g ow h and sp ead [ 20 ]. The ea ly and ma ked up egula ion o VCAM- 1 makes his molecule a po en ial ea ly bioma ke o de ec ing me as a ic ac i i y, o e ing ad an ages o e adi ional imaging ma ke s ha may no clea ly dis inguish be ween umou s ages o iden i y ea ly me as a ic sp ead. To his end, using an MRI con as agen based on mic opa icles o i on oxide (MPIO) and a ge ed o VCAM-1, we ha e p e iously demons a ed ha VCAM-1 p o ides a sensi i e and speci ic endo helial bioma ke o ea ly b ain me as asis [14,15], as well as imp o ing de ec ion o he umou -b ain in e ace [21]. P e ious s udies ha e also shown ha VCAM-1 is ma kedly up egula ed in bo h lung and li e me as ases [ 22 , 23 ], wi h li le exp ession in he no mal lung endo helium. Thus, VCAM-1 po en ially ep esen s a a ge o he ea ly de ec ion o mic ome as asis in he lungs. Howe e , owing o he low p o on densi y o he lung issue, i is no possible o e ec i ely de ec he p esence o MPIO by MRI in he lung, as his elies on dephasing exis ing p o on signals o achie e binding-speci ic con as . Al e na i ely, posi on emission omog aphy (PET), which is ypically combined wi h CT, o e s an a ac i e solu ion as i p o ides high sensi i i y (10 −12 M), enabling he loca ion and dis ibu ion o adiolabelled molecules in he lung o be acked in i o . The p e ailing PET imaging agen o iden i ying pulmona y me as ases is 18 F-FDG, which accumula es in egions wi h ele a ed glucose me abolism. This unc ional imaging me hod, howe e , is size-limi ed o umou s g ea e han 1 cm in diame e [ 24 , 25 ]. In he cu en s udy, In . J. Mol. Sci. 2024,25, 7160 3 o 19 he e o e, we p opose ha labelling he VCAM-MPIO con as agen wi h a posi on- emi ing adioiso ope such as 89 Z will enable PET de ec ion o mic ome as ases in he lung. In p io s udies, PET p obes ea u ing di e en adioiso opes di ec ed owa ds VCAM-1 ha e been epo ed, u ilising ei he nanobodies de i ed om unique hea y-chain-only an ibodies [ 26 ] o an ibodies alone [ 27 ]. Howe e , hese p obes ha e e y long ci cula ion imes in he blood and a e also likely o ex a asa e om he ascula u e. Ou app oach main ains all he ad an ages o he MPIO-based s a egy, including sho ci cula o y hal - li e and high alency o a ge ing ligands, while enabling de ec ion in an o gan ha would be insensi i e o he molecula MRI app oach. Fu he , his no el agen would enable sequen ial o concu en MRI and PET de ec ion o mic ome as ases in di e en o gans (e.g., he b ain and lungs) wi hin he same subjec . The aim o his s udy, he e o e, was o de elop a dual-con as PET/MRI p obe a ge ing VCAM-1 o enable he ea ly de ec ion o mic ome as ases in bo h he b ain and lungs using a single con as agen . This dual-modali y app oach no only p omises o imp o e he accu acy o me as asis de ec ion bu may also acili a e he ea ly ini ia ion o a ge ed he apies, po en ially imp o ing pa ien ou comes signi ican ly. 2. Resul s 2.1. Non-Radiolabelled VCAM-, IgG- o BSA-MPIO Conjuga es 2.1.1. Syn hesis and Loading o Conjuga es Bo h VCAM-MPIO and IgG-MPIO we e success ully syn hesised, and he an ibody loading was calcula ed o be ca. 3350 an ibodies pe µ m 2 o he MPIO su ace (11,650 pe MPIO). Following he 89 Z adiolabelling eac ions, app oxima ely 250 89 Z a oms we e associa ed wi h each MPIO (MA = 1.06 ×10−4MBq/µg o MPIO). 2.1.2. Ex Vi o Quan i a ion o VCAM-MPIO Binding in Pulmona y Me as asis Model Adhe en VCAM-MPIO we e e iden in me as asis-associa ed mic o ascula u e and nea by la ge essels in he lungs o mice injec ed wi h VCAM-MPIO (Figu e 1A,B; Figu e S1 A), while ew o no MPIO we e ound in he lungs o naï e mice injec ed wi h VCAM-MPIO. Speci ic binding o VCAM-MPIO on ma ked VCAM-1 exp essing lung ascula u e was con i med immunohis ochemically (Figu e 1A,B) and, again, was absen om he lungs o naï e mice. S udies in an ini ial coho o animals (n = 6) demons a ed he ep oducibili y o he lung mic ome as asis model used, alida ing subsequen com- pa isons be ween g oups; no signi ican di e ences we e ound in ei he umou numbe ( 4.50 ±1.23 ; mean numbe o me as ases pe lung sec ion analysed ± s anda d de ia- ion [SD]) o a ea-de i ed diame e (137.01 ± 45.17 µ m; mean ± SD) be ween animals (Figu e S1B,C). A signi ican ly g ea e numbe o VCAM-MPIO han IgG-MPIO (quan i ied as he numbe o adhe en MPIO pe µ m o in lamed endo helium, excluding capilla ies) we e ound associa ed wi h mic ome as a ic colonies a 10 min pos -injec ion [VCAM-MPIO s. IgG-MPIO, (9.80 ± 1.35) × 10 −3 s. (4.45 ± 4.08) × 10 −4 MPIO/ µ m endo helium; mean ± SD; unpai ed es p< 0.005; Figu e 1C]. VCAM-MPIO e en ion in me as asis- bea ing mice was signi ican ly educed a 3 h compa ed o me as asis-bea ing mice a 10 min pos -injec ion [(1.54 ± 1.82) × 10 −3 s. (9.8 ± 1.35) × 10 −3 MPIO/ µ m 2 umou a ea; mean ±SD ;p< 0.01; Figu e 1C], mos likely owing o immune cell (neu ophil) phagocy osis (Figu e S2A,B). Quan i a i e analysis o non-speci ic MPIO e en ion assessed om no mal issue (i.e., no con aining me as ases) in he lungs o 4T1-GFP injec ed animals showed subs an ially lowe le els han o me as asis-associa ed e en ion a 10 min pos -injec ion ((2.51 ± 1.07) × 10 −5 MPIO/ µ m 2 s. (0.12 ± 0.09) × 10 −2 MPIO/ µ m 2 ,p< 0.0001; Figu e 1D). Simila ly, low le els o e en ion we e ound in he naï e mice injec ed wi h ei he VCAM-MPIO ((1.22 ±0.05) ×10−5MPIO/µm2) o IgG-MPIO ((0.64 ±0.21) ×10−5MPIO/µm2). In . J. Mol. Sci. 2024,25, 7160 4 o 19 In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 o 19 Figu e 1. Quan i a i e and spa ial analysis o VCAM-MPIO and IgG-MPIO binding in lung issues o in me as asis-bea ing and naï e mice. (A,B) Rep esen a i e images o lung issue acqui ed om he me as a ic mouse model (day 10), 10 min pos -in a enous injec ion o VCAM-MPIO, s ained o VCAM-1 (b own) wi h a nuclea coun e s ain (blue). (A) Subs an ial numbe s o MPIO a e asso- cia ed wi h he me as asis mic o ascula u e ( ed a ows and highe magni ica ion a eas), and also on nea by la ge essels showing high VCAM-1 exp ession (B). (C,D) Immunohis ochemical quan- i a ion o MPIO binding in me as asis-bea ing o naï e lungs (10 min o 3 h a e i. . injec ion). (C) Ba g aphs ep esen he numbe o MPIO pe µm essel ci cum e ence and (D) he numbe o MPIO pe µm 2 in a eas o me as asis (me ), a eas o no appa en me as asis in he me as asis model (no me ), o heal hy lung issue (naï e mice) 10 min a e i. . injec ion. A 10 min pos -adminis a ion VCAM-MPIO showed signi ican ly g ea e e en ion on VCAM-1 exp essing essels associa ed wi h mic ome as ases compa ed o IgG-MPIO con ol). Simila le els o a ge ed and con ol agen e- en ion a 3 h indica e apid clea ance o MPIO om he issue. * p < 0.01; ** p < 0.005 compa ed o VCAM-MPIO e en ion a 10 min pos -injec ion. Non-speci ic e en ion was negligible compa ed o speci ic binding. A signi ican ly g ea e numbe o VCAM-MPIO han IgG-MPIO (quan i ied as he numbe o adhe en MPIO pe µm o in lamed endo helium, excluding capilla ies) we e ound associa ed wi h mic ome as a ic colonies a 10 min pos -injec ion [VCAM-MPIO s. IgG-MPIO, (9.80 ± 1.35) × 10 −3 s. (4.45 ± 4.08) × 10 −4 MPIO/µm endo helium; mean ± SD; unpai ed es p < 0.005; Figu e 1C]. VCAM-MPIO e en ion in me as asis-bea ing mice was signi ican ly educed a 3 h compa ed o me as asis-bea ing mice a 10 min pos -in- jec ion [(1.54 ± 1.82) × 10 −3 s. (9.8 ± 1.35) × 10 −3 MPIO/µm 2 umou a ea; mean ± SD; p < Figu e 1. Quan i a i e and spa ial analysis o VCAM-MPIO and IgG-MPIO binding in lung issues o in me as asis-bea ing and naï e mice. (A,B) Rep esen a i e images o lung issue acqui ed om he me as a ic mouse model (day 10), 10 min pos -in a enous injec ion o VCAM-MPIO, s ained o VCAM-1 (b own) wi h a nuclea coun e s ain (blue). (A) Subs an ial numbe s o MPIO a e associa ed wi h he me as asis mic o ascula u e ( ed a ows and highe magni ica ion a eas), and also on nea by la ge essels showing high VCAM-1 exp ession (B). (C,D) Immunohis ochemical quan i a ion o MPIO binding in me as asis-bea ing o naï e lungs (10 min o 3 h a e i. . injec ion). (C) Ba g aphs ep esen he numbe o MPIO pe µ m essel ci cum e ence and (D) he numbe o MPIO pe µ m 2 in a eas o me as asis (me ), a eas o no appa en me as asis in he me as asis model (no me ), o heal hy lung issue (naï e mice) 10 min a e i. . injec ion. A 10 min pos - adminis a ion VCAM-MPIO showed signi ican ly g ea e e en ion on VCAM-1 exp essing essels associa ed wi h mic ome as ases compa ed o IgG-MPIO con ol). Simila le els o a ge ed and con ol agen e en ion a 3 h indica e apid clea ance o MPIO om he issue. * p< 0.01; ** p< 0.005 compa ed o VCAM-MPIO e en ion a 10 min pos -injec ion. Non-speci ic e en ion was negligible compa ed o speci ic binding. 2.2. Radiolabelled VCAM-, IgG- o BSA-MPIO Conjuga es In Vi o Binding o [89Z ]-DFO-VCAM-MPIO Radioimmunoassays we e pe o med o con i m ha he binding cha ac e is ics o VCAM-MPIO we e no a ec ed by adiolabelling, and indica ed signi ican ly g ea e binding o [ 89 Z ]-DFO-VCAM-MPIO o ecombinan mouse VCAM-1 (% binding ± SD, 29.51 ± 7.03; Figu e 2A) han bo h o he nega i e con ols [ 89 Z ]-DFO-IgG-MPIO (10.34 ± 0.57; In . J. Mol. Sci. 2024,25, 7160 5 o 19 p< 0.001) and [ 89 Z ]-DFO (13.41 ± 1.20; p< 0.005). No signi ican di e ence was ound be ween he binding o [ 89 Z ]-DFO-VCAM-MPIO and he posi i e con ol [ 89 Z ]-DFO- VCAM (22.78 ± 2.51). A signi ican educ ion in binding was obse ed ollowing pa ial blocking wi h VCAM-1 an ibody (% cpm ± SD, 14.30 ± 2.28; p< 0.001 compa ed o unblocked binding; Figu e 2A). In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 5 o 19 0.01; Figu e 1C], mos likely owing o immune cell (neu ophil) phagocy osis (Figu e S2A,B). Quan i a i e analysis o non-speci ic MPIO e en ion assessed om no mal issue (i.e., no con aining me as ases) in he lungs o 4T1-GFP injec ed animals showed subs an- ially lowe le els han o me as asis-associa ed e en ion a 10 min pos -injec ion ((2.51 ± 1.07) × 10 −5 MPIO/µm 2 s. (0.12 ± 0.09) × 10 −2 MPIO/µm 2 , p < 0.0001; Figu e 1D). Simila ly, low le els o e en ion we e ound in he naï e mice injec ed wi h ei he VCAM-MPIO ((1.22 ± 0.05) × 10 −5 MPIO/µm 2 ) o IgG-MPIO ((0.64 ± 0.21) × 10 −5 MPIO/µm 2 ). 2.2. Radiolabelled VCAM-, IgG- o BSA-MPIO Conjuga es In Vi o Binding o [ 89 Z ]-DFO-VCAM-MPIO Radioimmunoassays we e pe o med o con i m ha he binding cha ac e is ics o VCAM-MPIO we e no affec ed by adiolabelling, and indica ed signi ican ly g ea e binding o [ 89 Z ]-DFO-VCAM-MPIO o ecombinan mouse VCAM-1 (% binding ± SD, 29.51 ± 7.03; Figu e 2A) han bo h o he nega i e con ols [ 89 Z ]-DFO-IgG-MPIO (10.34 ± 0.57; p < 0.001) and [ 89 Z ]-DFO (13.41 ± 1.20; p < 0.005). No signi ican diffe ence was ound be ween he binding o [ 89 Z ]-DFO-VCAM-MPIO and he posi i e con ol [ 89 Z ]-DFO- VCAM (22.78 ± 2.51). A signi ican educ ion in binding was obse ed ollowing pa ial blocking wi h VCAM-1 an ibody (% cpm ± SD, 14.30 ± 2.28; p < 0.001 compa ed o un- blocked binding; Figu e 2A). Figu e 2. Speci ici y and compe i i e inhibi ion o [ 89 Z ]-DFO-VCAM-MPIO binding o VCAM-1 and TNF-s imula ed HUVECs: adioimmunoassay e alua ion. (A) Radioimmunoassay plo s show- ing he % o bound adiolabelled agen s agains ecombinan VCAM-1 o (B) TNF-s imula ed HU- VECs wi h 30 min s a ic incuba ion, +/− compe i ion wi h VCAM-1 an ibody (blocking). (A) [ 89 Z ]- DFO-VCAM-MPIO showed signi ican ly g ea e % binding compa ed o bo h nega i e con ols [ 89 Z ]-DFO-IgG-MPIO and [ 89 Z ]-DFO (p < 0.005). Following blockade wi h VCAM-1 an ibody, [ 89 Z ]-DFO-VCAM-MPIO binding was signi ican ly educed (p < 0.001). (B) [ 89 Z ]-DFO-VCAM- MPIO showed signi ican ly g ea e % binding o TNF-s imula ed HUVECs compa ed o bo h nega- i e con ols [ 89 Z ]-DFO-IgG-MPIO and [ 89 Z ]-DFO (p < 0.001). Binding was subs an ially educed wi h he inclusion o he blocking an ibody and was negligible on uns imula ed cells. One-way ANOVA wi h Tukey pos -hoc es s; * p < 0.01; ** p < 0.005; *** p < 0.001; all compa isons a e agains [ 89 Z ]-DFO-VCAM-MPIO. Subsequen ly, binding o endo helial VCAM-1 was assessed on TNF-s imula ed HU- VECs and, again, [ 89 Z ]-DFO-VCAM-MPIO showed signi ican ly g ea e binding (% ± SD, 14.23 ± 4.43; p < 0.01; Figu e 2B) han ei he o he nega i e con ols [ 89 Z ]-DFO-IgG-MPIO (4.22 ± 0.92) and [ 89 Z ]-DFO (3.27 ± 0.72). In con as , he posi i e con ol [ 89 Z ]-DFO- VCAM showed simila le els o binding (17.69 ± 4.71) o [ 89 Z ]-DFO-VCAM-MPIO. P e- incuba ion wi h an excess o unlabelled VCAM-1 an ibody effec i ely ou -compe ed bind- ing o bo h [ 89 Z ]-DFO-VCAM-MPIO and [ 89 Z ]-DFO-VCAM o TNF-s imula ed HUVECs (% cpm ± SD, 4.45 ± 0.94 and 6.93 ± 0.99, espec i ely; Figu e 2B). The binding o [ 89 Z ]- DFO-VCAM-MPIO o naï e uns imula ed HUVECs was negligible (1.59 ± 0.41; Figu e 2B). Figu e 2. Speci ici y and compe i i e inhibi ion o [ 89 Z ]-DFO-VCAM-MPIO binding o VCAM-1 and TNF-s imula ed HUVECs: adioimmunoassay e alua ion. (A) Radioimmunoassay plo s showing he % o bound adiolabelled agen s agains ecombinan VCAM-1 o (B) TNF-s imula ed HUVECs wi h 30 min s a ic incuba ion, +/ − compe i ion wi h VCAM-1 an ibody (blocking). (A) [ 89 Z ]- DFO-VCAM-MPIO showed signi ican ly g ea e % binding compa ed o bo h nega i e con ols [ 89 Z ]-DFO-IgG-MPIO and [ 89 Z ]-DFO (p< 0.005). Following blockade wi h VCAM-1 an ibody, [ 89 Z ]-DFO-VCAM-MPIO binding was signi ican ly educed (p< 0.001). (B) [ 89 Z ]-DFO-VCAM- MPIO showed signi ican ly g ea e % binding o TNF-s imula ed HUVECs compa ed o bo h nega i e con ols [ 89 Z ]-DFO-IgG-MPIO and [ 89 Z ]-DFO (p< 0.001). Binding was subs an ially educed wi h he inclusion o he blocking an ibody and was negligible on uns imula ed cells. One-way ANOVA wi h Tukey pos -hoc es s; * p< 0.01; ** p< 0.005; *** p< 0.001; all compa isons a e agains [ 89 Z ]-DFO- VCAM-MPIO. Subsequen ly, binding o endo helial VCAM-1 was assessed on TNF-s imula ed HU- VECs and, again, [ 89 Z ]-DFO-VCAM-MPIO showed signi ican ly g ea e binding (% ± SD, 14.23 ± 4.43; p< 0.01; Figu e 2B) han ei he o he nega i e con ols [ 89 Z ]-DFO-IgG- MPIO (4.22 ± 0.92) and [ 89 Z ]-DFO (3.27 ± 0.72). In con as , he posi i e con ol [ 89 Z ]- DFO-VCAM showed simila le els o binding (17.69 ± 4.71) o [ 89 Z ]-DFO-VCAM-MPIO. P e-incuba ion wi h an excess o unlabelled VCAM-1 an ibody e ec i ely ou -compe ed binding o bo h [ 89 Z ]-DFO-VCAM-MPIO and [ 89 Z ]-DFO-VCAM o TNF-s imula ed HU- VECs (% cpm ± SD, 4.45 ± 0.94 and 6.93 ± 0.99, espec i ely; Figu e 2B). The binding o [ 89 Z ]-DFO-VCAM-MPIO o naï e uns imula ed HUVECs was negligible (1.59 ± 0.41; Figu e 2B). 2.3. In Vi o Imaging 2.3.1. In Vi o PET Imaging o [89Z ]-DFO-VCAM-MPIO in Pulmona y Me as asis Model The binding o [ 89 Z ]-DFO-VCAM-MPIO in i o in mice injec ed wi h 4T1-GFP cells was compa ed o ha o bo h [ 89 Z ]-DFO-IgG-MPIO and he agen wi hou MPIO, [ 89 Z ]-DFO-VCAM, a 10 min pos -adminis a ion using PET. In acco d wi h he known ou e o MPIO clea ance, he highes le els o ac i i y o bo h [ 89 Z ]-DFO-VCAM-MPIO ( Figu es 3A and S3A ) and [ 89 Z ]-DFO-IgG-MPIO (Figu e 3B and Figu e S3B) we e ound in he li e and spleen. Al hough adioac i i y wi hin he lungs was no isually appa en on indi idual single-slice s a ic PET images om pulmona y me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (Figu e S2A,B), maximum in ensi y plo s ob ained om he ull 3D da ase e ealed ma ked adioac i e up ake in he lungs (Figu e 3A) compa ed o me as asis-bea ing mice injec ed wi h he iso ype con ol agen [ 89 Z ]-DFO-IgG-MPIO (Figu e 3B). Al hough adioac i i y was clea ly e iden in he 3D maximum in ensi y plo s In . J. Mol. Sci. 2024,25, 7160 6 o 19 om he diseased lungs, his could no be esol ed in o indi idual mic ome as a ic colonies (Figu e 3A), likely owing o a combina ion o mo emen and he ela i ely low spa ial esolu ion o he PET images. In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 o 19 2.3. In Vi o Imaging 2.3.1. In Vi o PET Imaging o [ 89 Z ]-DFO-VCAM-MPIO in Pulmona y Me as asis Model The binding o [ 89 Z ]-DFO-VCAM-MPIO in i o in mice injec ed wi h 4T1-GFP cells was compa ed o ha o bo h [ 89 Z ]-DFO-IgG-MPIO and he agen wi hou MPIO, [ 89 Z ]- DFO-VCAM, a 10 min pos -adminis a ion using PET. In acco d wi h he known ou e o MPIO clea ance, he highes le els o ac i i y o bo h [ 89 Z ]-DFO-VCAM-MPIO (Figu es 3A and S3A) and [ 89 Z ]-DFO-IgG-MPIO (Figu es 3B and S3B) we e ound in he li e and spleen. Al hough adioac i i y wi hin he lungs was no isually appa en on indi idual single-slice s a ic PET images om pulmona y me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (Figu e S2A,B), maximum in ensi y plo s ob ained om he ull 3D da ase e ealed ma ked adioac i e up ake in he lungs (Figu e 3A) compa ed o me- as asis-bea ing mice injec ed wi h he iso ype con ol agen [ 89 Z ]-DFO-IgG-MPIO (Fig- u e 3B). Al hough adioac i i y was clea ly e iden in he 3D maximum in ensi y plo s om he diseased lungs, his could no be esol ed in o indi idual mic ome as a ic colo- nies (Figu e 3A), likely owing o a combina ion o mo emen and he ela i ely low spa ial esolu ion o he PET images. Figu e 3. Diffe en ial up ake o adiolabelled VCAM-MPIO and IgG-MPIO in me as asis-bea ing and naï e mice. (A,B) In i o, non-ga ed maximum in ensi y plo s ob ained om 3D PET da ase s om mice wi h pulmona y me as ases and injec ed wi h (A) [ 89 Z ]-DFO-VCAM-MPIO o (B) [ 89 Z ]- DFO-IgG-MPIO. (A) Ma ked adioac i i y is e iden in he lungs, li e , and spleen in he me as asis- bea ing mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO, bu (B) only om he li e and spleen in he me as asis-bea ing mouse injec ed wi h he iso ype con ol agen [ 89 Z ]-DFO-IgG-MPIO. (C,D) Rep- esen a i e a e age ac i i y up ake cu es o (C) [ 89 Z ]-DFO-VCAM-MPIO and (D) [ 89 Z ]-DFO- IgG-MPIO in li e , blood, and lungs om me as asis-bea ing mice (n = 4 pe g oup). The li e shows apid and high accumula ion o adioac i i y in bo h a ge ed and con ol g oups. (C) Lungs showed a highe accumula ion o ac i i y o he [ 89 Z ]-DFO-VCAM-MPIO injec ed mice han (D) he [ 89 Z ]- DFO-IgG-MPIO g oup. (E–G) G aphs showing a e age ac i i y up ake o e a du a ion o 55 min o li e , blood and lung om me as asis-bea ing mice injec ed wi h (E) [ 89 Z ]-DFO-VCAM-MPIO o (F) [ 89 Z ]-DFO-IgG-MPIO, o (G) om naï e mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO. In all cases, he li e showed signi ican ly highe up ake han o he issues (p < 0.0001), e lec ing he ole o he li e in sca enging unbound MPIO om he blood. (E) Lungs om me as asis-bea ing mice injec ed wi h he a ge ed agen showed signi ican ly (p < 0.0005) highe up ake han (F) lungs om mice injec ed wi h he con ol agen o (G) lungs om naï e mice injec ed wi h he a ge ed agen (p < 0.0005). Figu e 3. Di e en ial up ake o adiolabelled VCAM-MPIO and IgG-MPIO in me as asis-bea ing and naï e mice. (A,B) In i o , non-ga ed maximum in ensi y plo s ob ained om 3D PET da ase s om mice wi h pulmona y me as ases and injec ed wi h (A) [ 89 Z ]-DFO-VCAM-MPIO o (B) [ 89 Z ]- DFO-IgG-MPIO. (A) Ma ked adioac i i y is e iden in he lungs, li e , and spleen in he me as asis- bea ing mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO, bu (B) only om he li e and spleen in he me as asis-bea ing mouse injec ed wi h he iso ype con ol agen [ 89 Z ]-DFO-IgG-MPIO. (C,D) Rep esen a i e a e age ac i i y up ake cu es o (C) [ 89 Z ]-DFO-VCAM-MPIO and (D) [ 89 Z ]- DFO-IgG-MPIO in li e , blood, and lungs om me as asis-bea ing mice (n = 4 pe g oup). The li e shows apid and high accumula ion o adioac i i y in bo h a ge ed and con ol g oups. (C) Lungs showed a highe accumula ion o ac i i y o he [ 89 Z ]-DFO-VCAM-MPIO injec ed mice han (D) he [ 89 Z ]-DFO-IgG-MPIO g oup. (E–G) G aphs showing a e age ac i i y up ake o e a du a ion o 55 min o li e , blood and lung om me as asis-bea ing mice injec ed wi h (E) [ 89 Z ]-DFO-VCAM- MPIO o (F) [ 89 Z ]-DFO-IgG-MPIO, o (G) om naï e mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO. In all cases, he li e showed signi ican ly highe up ake han o he issues (p< 0.0001), e lec ing he ole o he li e in sca enging unbound MPIO om he blood. (E) Lungs om me as asis-bea ing mice injec ed wi h he a ge ed agen showed signi ican ly (p< 0.0005) highe up ake han (F) lungs om mice injec ed wi h he con ol agen o (G) lungs om naï e mice injec ed wi h he a ge ed agen (p< 0.0005). Naï e (con ol) mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO also showed up ake, p ima ily in he li e and spleen (Figu e S2C). Me as asis-bea ing animals injec ed wi h [ 89 Z ]-DFO-VCAM (an ibody alone) showed high le els o he agen in he blood o he i s hou (Figu e S2D,E) and high le els o adioac i i y in he hea . The li e and bladde also showed high up ake o adioac i i y, whe eas lowe le els o adioac i i y we e e iden in he spleen and bones (Figu e S2D). Despi e he lack o isible ocal signal in he lungs o me as asis-bea ing animals injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO, ime-ac i i y cu es o he lungs showed clea ac i i y up ake (equilib a ing o 0.53 ± 0.28 g/mL; mean ± SD; Figu e 3C,E). This up ake was signi ican ly highe han he ac i i y up ake o ei he [ 89 Z ]-DFO-IgG-MPIO in lungs om me as asis-bea ing mice (0.12 ± 0.04 g/mL; mean ± SD; p< 0.0001; Figu e 3D,F) o [ 89 Z ]-DFO-VCAM-MPIO up ake in naï e mouse lungs (0.10 ± 0.02 g/mL; mean ± SD; In . J. Mol. Sci. 2024,25, 7160 7 o 19 p< 0.0001; Figu e 3G). The blood ac i i y in all g oups was consis en ly lowe han ha o lung up ake o [ 89 Z ]-DFO-VCAM-MPIO in me as asis-bea ing animals ( Figu e 3C–G ; p< 0.0001). Time-ac i i y cu es o he li e s o me as asis-bea ing animals injec ed wi h [ 89 Z ]- DFO-VCAM-MPIO (Figu e 3C) o [ 89 Z ]-DFO-IgG-MPIO (Figu e 3D) showed a apid inc ease in up ake, which pla eaued a 5 min pos -adminis a ion. Me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO showed compa able le els o li e up ake, o e 50 min, o hose injec ed wi h ei he [ 89 Z ]-DFO-IgG-MPIO (2.65 ± 0.28 s. 2.38 ± 0.12 g/mL; mean ± SD; Figu e 3E,F) o naï e mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (2.52 ± 0.32 g/mL; mean ±SD; Figu e 3E,G). Ac i i y up ake cu es o [ 89 Z ]-DFO-VCAM in me as asis-bea ing mice showed a apid inc ease in up ake in o he hea , li e , and bladde (Figu e S2E), eaching a pla eau a 5 min pos -adminis a ion. The hal -li e o [ 89 Z ]-DFO-VCAM in he ci cula ion was de- e mined om he hea measu emen s. Hea measu emen s we e also used o de e mine he hal -li e o [ 89 Z ]-DFO-VCAM-MPIO o [ 89 Z ]-DFO-IgG-MPIO in he ci cula ion, and indings we e consis en wi h he alues calcula ed om he biological clea ance s udy; ci cula o y hal -li e PET s. MRI, 36.6 s. 39.9 s (Figu e S3). S a ic analysis o lung ac i i y was also pe o med a wo ime poin s, 10 and 55 min pos -adminis a ion, on he 1 h in i o dynamic da ase . Lungs om he me as asis-bea ing g oup injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO showed signi ican ly g ea e (p< 0.0005) ac i i y compa ed o bo h me as asis-bea ing animals injec ed wi h [ 89 Z ]-DFO-IgG-MPIO and naï e animals injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO a bo h ime poin s (Figu e 4A,B). In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 o 19 Naï e (con ol) mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO also showed up ake, p ima ily in he li e and spleen (Figu e S2C). Me as asis-bea ing animals injec ed wi h [ 89 Z ]-DFO-VCAM (an ibody alone) showed high le els o he agen in he blood o he i s hou (Figu e S2D,E) and high le els o adioac i i y in he hea . The li e and bladde also showed high up ake o adioac i i y, whe eas lowe le els o adioac i i y we e e i- den in he spleen and bones (Figu e S2D). Despi e he lack o isible ocal signal in he lungs o me as asis-bea ing animals in- jec ed wi h [ 89 Z ]-DFO-VCAM-MPIO, ime-ac i i y cu es o he lungs showed clea ac- i i y up ake (equilib a ing o 0.53 ± 0.28 g/mL; mean ± SD; Figu e 3C,E). This up ake was signi ican ly highe han he ac i i y up ake o ei he [ 89 Z ]-DFO-IgG-MPIO in lungs om me as asis-bea ing mice (0.12 ± 0.04 g/mL; mean ± SD; p < 0.0001; Figu e 3D,F) o [ 89 Z ]- DFO-VCAM-MPIO up ake in naï e mouse lungs (0.10 ± 0.02 g/mL; mean ± SD; p< 0.0001; Figu e 3G). The blood ac i i y in all g oups was consis en ly lowe han ha o lung up- ake o [ 89 Z ]-DFO-VCAM-MPIO in me as asis-bea ing animals (Figu e 3C–G; p < 0.0001). Time-ac i i y cu es o he li e s o me as asis-bea ing animals injec ed wi h [ 89 Z ]- DFO-VCAM-MPIO (Figu e 3C) o [ 89 Z ]-DFO-IgG-MPIO (Figu e 3D) showed a apid in- c ease in up ake, which pla eaued a 5 min pos -adminis a ion. Me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO showed compa able le els o li e up ake, o e 50 min, o hose injec ed wi h ei he [ 89 Z ]-DFO-IgG-MPIO (2.65 ± 0.28 s. 2.38 ± 0.12 g/mL; mean ± SD; Figu e 3E,F) o naï e mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (2.52 ± 0.32 g/mL; mean ± SD; Figu e 3E,G). Ac i i y up ake cu es o [ 89 Z ]-DFO-VCAM in me as asis-bea ing mice showed a apid inc ease in up ake in o he hea , li e , and bladde (Figu e S2E), eaching a pla eau a 5 min pos -adminis a ion. The hal -li e o [ 89 Z ]-DFO-VCAM in he ci cula ion was de- e mined om he hea measu emen s. Hea measu emen s we e also used o de e mine he hal -li e o [ 89 Z ]-DFO-VCAM-MPIO o [ 89 Z ]-DFO-IgG-MPIO in he ci cula ion, and indings we e consis en wi h he alues calcula ed om he biological clea ance s udy; ci cula o y hal -li e PET s. MRI, 36.6 s. 39.9 s (Figu e S3). S a ic analysis o lung ac i i y was also pe o med a wo ime poin s, 10 and 55 min pos -adminis a ion, on he 1 h in i o dynamic da ase . Lungs om he me as asis-bea - ing g oup injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO showed signi ican ly g ea e (p < 0.0005) ac i i y compa ed o bo h me as asis-bea ing animals injec ed wi h [ 89 Z ]-DFO-IgG-MPIO and naï e animals injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO a bo h ime poin s (Figu e 4A,B). Figu e 4. Tempo al and spa ial analysis o adiolabelled VCAM-MPIO o IgG-MPIO up ake in me as asis-bea ing and naï e mice. (A,B) In i o a e age ac i i y up ake in he lungs o mice injec ed wi h (A) [ 89 Z ]-DFO-VCAM-MPIO a 10 and (B) 50 min pos -adminis a ion (n = 4 pe g oup) showed signi ican ly g ea e e en ion in me as asis-bea ing lungs compa ed o naï e lungs o o me as asis- bea ing mice injec ed wi h [ 89 Z ]-DFO-IgG-MPIO (*** p< 0.0001). (C,D) Pos -mo em (10 min pos i. .) 2 h acquisi ion whole-body PET images o mice injec ed wi h (C) [ 89 Z ]-DFO-VCAM-MPIO o (D) [ 89 Z ]-DFO-IgG-MPIO showing ma ked adioac i i y in he li e and spleen. (C) Lungs om he mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO also show no able adioac i i y (D), whe eas signals om he lungs o he mouse injec ed wi h [ 89 Z ]-DFO-IgG-MPIO do no . (E) The mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO in image (C) was u he examined using a long PET acquisi ion (60 h) a e emo ing he body and o gans below he diaph agm, o con i m ha he appa en lung signal, as seen in image (C), o igina ed om he lungs and was no a pa ial olume e ec om li e and spleen. In . J. Mol. Sci. 2024,25, 7160 8 o 19 2.3.2. Pos Mo em PET Imaging o [89Z ]-DFO-VCAM-MPIO in Pulmona y Me as asis Model To ci cum en issues o mo ion in he in i o PET images, wo me as asis-bea ing mice injec ed wi h ei he [ 89 Z ]-DFO-VCAM-MPIO (Figu e 4C) o [ 89 Z ]-DFO-IgG-MPIO (Figu e 4D) we e e mina ed 10 min pos -adminis a ion and imaged o e a 2 h ime ame wi h PET. Bo h images showed high le els o adioac i i y in he li e , while only he mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO showed signi ican adioac i i y in he lungs. Subsequen ly, he mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO was u he in es iga ed wi h 60 h PET acquisi ion a e excision o all o gans below he diaph agm o e i y ha he signal o igina ed om he lungs and was no a pa ial olume e ec om he li e and spleen (Figu e 4E). 2.4. Ex Vi o Analysis 2.4.1. Ex Vi o Biodis ibu ion o [89Z ]-DFO-VCAM-MPIO Biodis ibu ion o all agen s was u he assessed ex i o, and he highes up ake ac oss all o he o gans s udied was ound in he lungs om me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (68.9 ± 22.9% ID/g; mean ± SD); lung up ake was signi ican ly highe han ha in all o he expe imen al g oups (one-way ANOVA, p< 0.05; Figu e 5). The spleen and li e exhibi ed he second highes ac i i y up ake in me as asis- bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (49.6 ± 37.6% ID/g; mean ± SD), and he le els we e simila in all g oups. The hea , kidney, bone, and blood samples showed ela i ely low le els o up ake, while he s omach, small and la ge in es ine, panc eas, and samples om muscle, skin, and a showed negligible up ake in all g oups (Figu e 5). In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 o 19 Figu e 4. Tempo al and spa ial analysis o adiolabelled VCAM-MPIO o IgG-MPIO up ake in me- as asis-bea ing and naï e mice. (A,B) In i o a e age ac i i y up ake in he lungs o mice injec ed wi h (A) [ 89 Z ]-DFO-VCAM-MPIO a 10 and (B) 50 min pos -adminis a ion (n = 4 pe g oup) showed signi ican ly g ea e e en ion in me as asis-bea ing lungs compa ed o naï e lungs o o me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO-IgG-MPIO (*** p < 0.0001). (C,D) Pos -mo em (10 min pos i. .) 2 h acquisi ion whole-body PET images o mice injec ed wi h (C) [ 89 Z ]-DFO-VCAM- MPIO o (D) [ 89 Z ]-DFO-IgG-MPIO showing ma ked adioac i i y in he li e and spleen. (C) Lungs om he mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO also show no able adioac i i y (D), whe eas signals om he lungs o he mouse injec ed wi h [ 89 Z ]-DFO-IgG-MPIO do no . (E) The mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO in image (C) was u he examined using a long PET acquisi ion (60 h) a e emo ing he body and o gans below he diaph agm, o con i m ha he appa en lung signal, as seen in image (C), o igina ed om he lungs and was no a pa ial olume effec om li e and spleen. 2.3.2. Pos Mo em PET Imaging o [ 89 Z ]-DFO-VCAM-MPIO in Pulmona y Me as asis Model To ci cum en issues o mo ion in he in i o PET images, wo me as asis-bea ing mice injec ed wi h ei he [ 89 Z ]-DFO-VCAM-MPIO (Figu e 4C) o [ 89 Z ]-DFO-IgG-MPIO (Figu e 4D) we e e mina ed 10 min pos -adminis a ion and imaged o e a 2 h ime ame wi h PET. Bo h images showed high le els o adioac i i y in he li e , while only he mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO showed signi ican adioac i i y in he lungs. Subsequen ly, he mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO was u he in- es iga ed wi h 60 h PET acquisi ion a e excision o all o gans below he diaph agm o e i y ha he signal o igina ed om he lungs and was no a pa ial olume effec om he li e and spleen (Figu e 4E). 2.4. Ex Vi o Analysis 2.4.1. Ex Vi o Biodis ibu ion o [ 89 Z ]-DFO-VCAM-MPIO Biodis ibu ion o all agen s was u he assessed ex i o, and he highes up ake ac oss all o he o gans s udied was ound in he lungs om me as asis-bea ing mice in- jec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (68.9 ± 22.9% ID/g; mean ± SD); lung up ake was signi ican ly highe han ha in all o he expe imen al g oups (one-way ANOVA, p < 0.05; Figu e 5). The spleen and li e exhibi ed he second highes ac i i y up ake in me as asis- bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (49.6 ± 37.6% ID/g; mean ± SD), and he le els we e simila in all g oups. The hea , kidney, bone, and blood samples showed ela i ely low le els o up ake, while he s omach, small and la ge in es ine, panc eas, and samples om muscle, skin, and a showed negligible up ake in all g oups (Figu e 5). Figu e 5. O gan-speci ic biodis ibu ion o adiolabelled VCAM-MPIO o IgG-MPIO in me as asis- bea ing and naï e mice. Ba g aph showing he a e age % o injec ed dose pe mass o o gan o all g oups (n = 8 pe g oup). In he lungs, me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO-VCAM- MPIO ( ed) showed signi ican ly (p < 0.05) g ea e adia ion yield han ei he he con ol [ 89 Z ]-DFO- IgG-MPIO injec ed g oups (blue and cyan) o naï e mice injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (o ange). The li e and spleen showed he nex highes le els o adioac i i y up ake, suppo ing Figu e 5. O gan-speci ic biodis ibu ion o adiolabelled VCAM-MPIO o IgG-MPIO in me as asis- bea ing and naï e mice. Ba g aph showing he a e age % o injec ed dose pe mass o o gan o all g oups (n = 8 pe g oup). In he lungs, me as asis-bea ing mice injec ed wi h [ 89 Z ]-DFO- VCAM-MPIO ( ed) showed signi ican ly (p< 0.05) g ea e adia ion yield han ei he he con ol [ 89 Z ]-DFO-IgG-MPIO injec ed g oups (blue and cyan) o naï e mice injec ed wi h [ 89 Z ]-DFO- VCAM-MPIO (o ange). The li e and spleen showed he nex highes le els o adioac i i y up ake, suppo ing hei ole in he sca enging o non-bound pa icles in ci cula ion. The kidney and bone showed compa able le els o %ID/g o he blood and hea , and hus, hey a e likely o be backg ound ci cula ing le els. One-way ANOVA; * p< 0.05; ** p< 0.01. 2.4.2. Lung Au o adiog aphy o [89Z ]-DFO-VCAM-MPIO Accumula ion in Lungs The binding o [ 89 Z ]-labelled MPIO was u he con i med by compa ison o au o a- diog aphy wi h immunohis ochemical assessmen o VCAM-1 exp ession and he p esence o me as ases in he same sec ions (Figu e 6). In all sec ions om me as asis-bea ing mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO, a low le el o ac i i y was e iden ac oss he whole lung, in addi ion o mo e in ense da k oci in disc e e a eas o he lungs (Figu e 6A). The negligible ac i i y was e iden in sec ions om ei he me as asis-bea ing o naï e mice injec ed wi h [ 89 Z ]-DFO-IgG-MPIO (Figu e 6C,D) and om he naï e mouse injec ed wi h [ 89 Z ]-DFO-VCAM-MPIO (Figu e 6B). Lung issue sec ions we e also used o longe In . J. Mol. Sci. 2024,25, 7160 9 o 19 exposu e au o adiog aphy and subsequen VCAM-1 immunohis ochemis y, and he p es- ence o me as ases a he loca ion o he in ense da k spo s on he au o adiog aphy ilm was con i med; hese u he co ela ed wi h he p esence o [ 89 Z ]-DFO-VCAM-MPIO (Figu e 6E–G ; Figu e S4B(1–3)). A longe exposu es, i was appa en ha some da k spo s on he ilm we e no co ela ed wi h me as ases bu we e co ela ed wi h he p esence o MPIO (Figu e S5B; small ed do s on he issue). These MPIO may be associa ed wi h me as ases in subsequen issue sec ions o may e lec non-speci ic e en ion owing o essel/ issue a chi ec u e. I was also ound ha occasional me as ases wi h a e y low MPIO sco e did no co ela e wi h oci (Figu e S4B(4)), mos likely owing o low 89 Z le els. Con e sely, in a small numbe o me as ases showing high exp ession o VCAM-1, i appea ed ha no MPIO we e p esen , bu hese s ill co ela ed wi h mode a e ac i i y oci (Figu e S5B(5)). In his case, i is possible ha he MPIO we e dislodged du ing he s aining p ocess o obscu ed by he e y in ense VCAM-1 s aining. The au o adiog aphy in Figu e 6E was pe o med a day ea lie han ha in Figu e 6A and was exposed o 24 h compa ed o 10 h o he sec ions in Figu e 6A. Because he exposu e ime was no p opo ional o he decay o he iso ope, he au o adiog aph in Figu e 6E appea s da ke han he samples in Figu e 6A, due o o e exposu e. P io o au o adiog aphy, he lungs we e no pe used wi h saline o emo e he blood om he ascula u e, and i is likely, he e o e, ha he unco ela ed da k spo s e lec an unknown amoun o ee 89 Z in he blood. This no ion is suppo ed by he highe signal obse ed in he hea , which con ains a g ea e blood olume han lung issue. I should also be no ed ha in some a eas whe e he issue is olded, a highe sa u a ed signal is e iden ha does no e lec he p esence o me as ases (Figu e 6E,F; Figu e S5). In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 10 o 19 Figu e 6. Compa a i e au o adiog aphic and immunohis ochemical analyses and colocalisa ion o oci wi h adiolabelled VCAM-MPIO and mic ome as ases. (A–D) Pai ed images o lung issue au- o adiog aphic da a (le ; 10 h exposu e) and b igh - ield mic og aphs o he associa ed lung sec ions ( igh ) s ained immunohis ochemically o VCAM-1 exp ession (b own) o (A) me as asis-bea ing o (B) naï e lungs ea ed wi h [ 89 Z ]-DFO-VCAM-MPIO; (C) me as asis-bea ing o (D) naï e lungs ea ed wi h [ 89 Z ]-DFO-IgG-MPIO. (A) Me as asis-bea ing lungs + [ 89 Z ]-DFO-VCAM-MPIO showed spa se basal ac i i y in all a eas o he lung sec ions and localised in ense signals (da k spo s). (B–D) All o he g oups showed no o minimal ac i i y. (E,F) Au o adiog aphic images om he lung issue sec ion a e 24 h exposu e and immunohis ochemical images om he same lung sec ion manually inspec ed unde magni ica ion. Foci o high ac i i y on he au o adiog aphic pla e (e.g., ed dashed squa e and ed ci cles) colocalise wi h VCAM-1 exp essing mic ome as a ic a eas (black dashed squa e and ed ci cles). The high ac i i y oci on he au o adiog aphic pla e ha do no co ela e wi h appa en me as ases do co ela e wi h a eas whe e MPIO we e manually sco ed ([F]; ha d o dis inguish small ed ma ks). Do ed scale ba = 5 mm. (G) Black dashed squa e in (F) is shown a a highe esolu ion; a ows indica e he p esence o MPIO. Nuclei a e s ained blue. Scale ba = 50 µm. 3. Discussion To da e, he e is no s anda d non-in asi e clinical o p eclinical sc eening me hod ha p o ides a diagnosis o ea ly mic ome as a ic de elopmen in he lung. In his s udy, we show ha [ 89 Z ]-DFO-VCAM-MPIO speci ically binds o ac i a ed pulmona y endo- helium a si es o mic ome as ases, enabling e y ea ly de ec ion o lung me as ases by PET imaging. The sp ead o me as a ic cells o he lympha ic sys em is common in many ypes o cance . Consequen ly, imaging o lymph nodes has become one o he s anda d clinical sc eening me hods o seconda y umou sp ead, and bo h s uc u al MRI [28] and nu- clea imaging wi h unc ional ace s [29,30] ha e enabled he de ec ion o malignan lymph nodes o many yea s. Con as -enhanced MRI using supe pa amagne ic pa icles o i on oxide (SPIO; 40– 150 nm) and ul asmall SPIO (USPIO; <50 nm) ha e been u ilised in he clinic o hei p ope ies o p e e en ially phagocy osed by cells in no mal lymphoid issues [31] and acili a ed a mo e accu a e assessmen o lymph node malignancy [32–36]. Howe e , hese me hods a e limi ed o he de ec ion o mac o- and mic ome as a ic lesions wi hin he lympha ic sys em and do no enable si e-speci ic iden i ica ion o sp ead o o he is- sues/o gans [16]. Ta ge ing o VCAM-1 wi h VCAM-MPIO and in i o MRI has p e iously been demons a ed in p eclinical models o nume ous diseases, including neu oin lamma ion Figu e 6. Compa a i e au o adiog aphic and immunohis ochemical analyses and colocalisa ion o oci wi h adiolabelled VCAM-MPIO and mic ome as ases. (A–D) Pai ed images o lung issue au o adiog aphic da a (le ; 10 h exposu e) and b igh - ield mic og aphs o he associa ed lung sec ions ( igh ) s ained immunohis ochemically o VCAM-1 exp ession (b own) o (A) me as asis- bea ing o (B) naï e lungs ea ed wi h [ 89 Z ]-DFO-VCAM-MPIO; (C) me as asis-bea ing o (D) naï e lungs ea ed wi h [ 89 Z ]-DFO-IgG-MPIO. (A) Me as asis-bea ing lungs + [ 89 Z ]-DFO-VCAM-MPIO showed spa se basal ac i i y in all a eas o he lung sec ions and localised in ense signals (da k spo s). (B–D) All o he g oups showed no o minimal ac i i y. (E,F) Au o adiog aphic images om he lung issue sec ion a e 24 h exposu e and immunohis ochemical images om he same lung sec ion manually inspec ed unde magni ica ion. Foci o high ac i i y on he au o adiog aphic pla e (e.g., ed In . J. Mol. Sci. 2024,25, 7160 16 o 19 MPIO o [ 89 Z ]-DFO-IgG-MPIO (0.5 MBq pe injec ion) and e mina ed wi h le hal injec ion (200 µ L pen oba bi al) 10 min la e (n = 8 pe g oup). Blood samples we e collec ed om each animal immedia ely a e incision in o he diaph agm by cu ing he in e io ena ca a and allowing he blood o pool wi hin he diaph agm-cons ained a ea. Subsequen ly, he hea , lungs, li e , spleen, s omach, la ge in es ine, small in es ine, panc eas, kidneys, and samples o bone ( igh ulna), skin (abdomen), and isce al a (abdomen) we e excised, washed in PBS ( o elimina e blood), and le o d y on high-abso bency pape . Selec ed o gans, issues, and blood we e placed in 5 mL ubes o known weigh (The mo Fishe Sci- en i ic, Wal ham, MA, USA), and he ubes we e placed in a gamma coun e ( 1470 WIZARD gamma coun e , Pe kin Elme , Wal ham, MA, USA) o de e mine adioac i i y pe sam- ple. Tubes con aining he o gans we e weighed o calcula e he mass o he o gan, and subsequen ly, he pe cen age o injec ed dose pe g (%ID/g) o he o gan was calcula ed. 4.5.2. Ex Vi o Au o adiog aphy o MPIO Accumula ion in Lungs G oups equi alen o hose desc ibed o he biodis ibu ion s udy we e used o ex i o au o adiog aphy (n = 2 pe g oup; 8 mice in o al). Lungs we e excised and ea ed as desc ibed abo e. The lungs we e kep in suc ose o 48 h and hen c yosec ioned as desc ibed abo e. A e c yosec ioning, he moun ed lung sec ions we e placed on a Cylcone ® s o age phospho sc een and exposed o 12 o 24 h a oom empe a u e. Subsequen ly, he same lung sec ions we e s ained immunohis ochemically o VCAM-1, as desc ibed abo e. 5. Conclusions In conclusion, we p esen he e a no el imaging me hod o he de ec ion o pulmona y mic ome as ases in mice by a ge ing ac i a ed pulmona y endo helium using a PET- de ec able molecula ly a ge ed con as agen , [ 89 Z ]-DFO-VCAM-MPIO. By keeping he mic opa icle pla o m used o ou molecula MRI con as agen s, we bo h e ain hei clea ance cha ac e is ics, yielding highly speci ic a ge signal changes and p o iding a dual-modali y agen ha can be used o de ec mic ome as ases in di e en issue beds wi h he op imal imaging app oach o each. The agen is in i sel a pla o m, as he basic MPIO componen can be conjuga ed o a ange o di e en a ge ing and con as molecules o c ea e a ied and mul i unc ional agen s. Looking ahead, he nex s eps o esea ch should ocus on u he e ining he p obe’s speci ici y and e iciency, leading o clinical ials. Downs eam, explo ing he in eg a ion o his imaging echnology in o ou ine clinical p ac ice will be c ucial. A he esea ch le el, expanding he applica ion o his p obe o o he ypes o cance could p o ide comp ehensi e insigh s in o i s e sa ili y and e ec i eness ac oss di e en oncological con ex s. These s eps will be pi o al in mo ing om expe imen al applica ion o a s anda d componen o oncological imaging and ea men planning, pa ing he way o ad ances in pe sonalised medicine and imp o ed p ognosis in pa ien s wi h cance . Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a h ps://www. mdpi.com/a icle/10.3390/ijms25137160/s1. Au ho Con ibu ions: N.R.S. and S.M. designed he expe imen s and p epa ed he manusc ip . S.M. and N.Z. pe o med he in i o wo k. J.C.K., B.C. and F.P.-B. designed and syn hesised he imaging agen . S.M., J.C.K. and V.K. pe o med he PET imaging and analysis. R.J.M. and N.R.S. supe ised he s udy, and M.S.S. p o ided essen ial supe ision and assis ance wi h his ology. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This wo k was unded by Cance Resea ch UK (g an numbe C5255/A15935) and he CR-UK & EPSRC Ox o d Cance Imaging Cen e (g an numbe C5255/A1646). SM was unded by a CR-UK & EPSRC Ox o d Cance Imaging Cen e S uden ship. Ins i u ional Re iew Boa d S a emen : All animal expe imen s we e app o ed by he Uni e si y o Ox o d Clinical Medicine E hics Re iew Commi ee and he UK Home O ice (Animals [Scien i ic P ocedu es] Ac , 1986). In . J. Mol. Sci. 2024,25, 7160 17 o 19 In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : The da ase s suppo ing he indings o he cu en s udy a e a ailable om he co esponding au ho upon eques . Acknowledgmen s: The au ho s hank he s a o he BMS acili y o assis ance wi h animal husband y and wel a e and membe s o he Ox o d Ins i u eImaging Co e (Sean Sma , Paul Kincesch, and Danny Allen) o imaging and analysis suppo . Con lic s o In e es : The wo k has no ele ance o Cu eVac and hey ha e no in e es in he ou come o he pape . Re e ences 1. Valas yan, S.; Weinbe g, R.A. Tumo Me as asis: Molecula Insigh s and E ol ing Pa adigms. Cell 2011,147, 275–292. [C ossRe ] [PubMed] 2. 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