Magnetic particle imaging reveals that iron-labeled extracellular vesicles accumulate in brains of mice with metastases

Ir on-lab eled EV s ha v e been imag ed pr ev iously using MRI 27–29,32,33 and MPI 34 . Methods used t o label EV s ha v e bee n thr ough di r ect la beling , or in dir ect lab eling where the pa r e nt al cell is c o-incubat ed with t he ir on and is allowed t o t ak e up the nano particle . In this s tu dy , w e f ound p r o t ami ne sulf at e and h epari n incr ease d pa r ent a l cell lab eling 101 , r esul ting in increased F eEV loading and imp r ov ed de t ec tion whe n used f or MPI. TEM imaging of F eEV s show s that ir on is associ a ted with t h e EV membr anes, and pr oteomics analy sis c onfirms tha t th e F e EV s c on t ain pr o teins that are c onside r ed EV mark er s. In primary br east tumor s in vivo, F eEV s fr om th e br ea s t cancer cells w e r e r e t aine d f or long er and in g r e a ter amoun ts as c ompared t o ir o n nanopa rticles alone , as demons tra ted using MPI and c onfirmed by his t ol ogic al analy sis of the tumor . Ass ocia tio n with EV membr anes also allow ed SPIO nanopar ticle deliv e ry t o th e heads of mice when br a in met ast ases w e r e p r ese n t . MPI c ould not de t ect iron in th e heads of he alth y mice injec t ed with FeEV s, nor in mic e with brain metast as es injected with SPIO nanopar ticles alone . W e c onclude th a t associa tion with EV membr anes allow ed f or the c ar go , ir on nanopar ticles, to access me t asta tic si t es acr oss th e BB B/B TB. In the future, they c ould ac t as a deliver y v ehicle f or therapeu tics associat ed with i r on nanop articl es or oth er th er ap eutic a g ents.

Cell Culture/iHJ 4T1fLuc2 cells (4T1L2; pr ovided by Dr . Bry an Smith, M SU), 4T1BGL cells (pr ovided by Dr . Michael Bachmann, MSU) and 4T1BR5-fLuc /GFP cells (4T1BR5- L2G; pr ovided by Dr . P aula F os t er , W estern Univ er sity) w e r e mai n tained i n incub a tors set at 37°C and 5% C O 2 . Cells wer e cul t ur ed in RPMI+ Glu t amax with 10% f etal bovine se rum (FBS). Cells w ere c o unt ed using th e T ryp an blue e x c lusion assa y prio r t o in vitro or i n vivo expe riments. Iron Lab eling of Cells Thr ee million cells w ere seeded in a 10 cm 2 dish f or ir on labeling. Pr ot amin e sulf a t e (40 µg /ml) or heparin (2 U/ml) and 70 nm dex tr an- c oat e d S ynomag-D (Micr oMod, Ge rma n y , c a t #104-00-701 or c a t #126-00-701 (f ar r ed fluorescence); 1 mg /ml F e) w er e add ed t o 2.5 ml of FBS-fr ee m edia. Both tub es w er e well mix ed , be f ore the protamine sulf a t e w as add ed to the hep arin and S yn omag-D . Fiv e ml of this mixtu r e w as added t o each plate and 3 t o 6-h la ter 5 ml of c omplet e media w as added. The cells w ere then incub at ed f or 24-h post-additi on of ir on prio r to F eEV isol a tion (below). The equiv ale n t of 1 dish of F eEV s w ere used f or i. v . biodis t ributi on and primary tumo r s tudi es and th e eq uiv alent of 2 dishes of F eEV s w ere used f or br ain metast asis s tu dies. F eEV Isolatio n Via Diff eren ti al Centrifug a tion .CC-BY-NC 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 2, 2024. ; https://doi.org/10.1101/2024.03.12.584146doi: bioRxiv preprint Ir on-lab eled cells were w ashed 3 tim es with 10 U/ml heparin and onc e with PBS, and r epl aced with media c o nt aining 10% EV-deple t ed F BS. Cells w ere incub a ted a t 37°C f or 24-h t o allow f or EV pr oduction . Conditioned me dia w as ce n trifuged a t 6 00g f or 10 min t o r emov e an y cells. The supern a tant w as th en cen t rifug ed at 2,000g f or 20 min t o r emov e apoptotic bodies and cell d ebris. The r emaining super nat a nt c ont aining F eEV s w as subsequently centrifug ed at 20,000g f or 1-h t o c oncentrat e F eEV s, which w er e then w ash ed in PBS an d furth er concent r at ed via ce n t rifug a tio n at 20,000g f or 1-h, be f ore resuspendi ng in PBS. EV Isolatio n via Diff erential Ul trace n trifu gati on Non-ir on l abele d 4T1 cells w e r e s eede d a t a densi ty of 3x10 6 cells in a 10 cm d ish. T w e n ty f our ho ur s aft er s eeding , the c ells w e r e w ashed twi ce with PBS t o r emove traces of med ia a nd r epl aced wi th medi a c ont aining 10% EV-deplet ed FBS. Cells w er e incub at ed at 37°C f or 24-h t o allow f or EV pr oduction . Conditioned med ia w as centrifuged at 600g f or 10 min t o r emov e an y cells an d the supe rnat a n t w as cen t rifug ed at 2,000g f or 20 min t o r e mov e apopt o tic bodies and cell debris. Supern at ant c ont aini ng non-labeled EV s w as r emov e d and ce n t r ifug ed at 100,000g f or 90 min t o c once n trat e non-la beled EV s . These non-lab eled EV s w e r e then washe d with PBS and recentrifuged f or puri ty be f or e r esusp ension i n PBS. F eEV or SPIO Upt ak e Into 4T1BR5-L2G Cells in Culture 200,000 4T1BR5-L2G cells w e r e s eede d per well, in a 2-w ell chamb ered sl ide (Nunc Lab-T ek II , c a t #S6565) and incuba ted a t 37°C and 5% C O 2 ov ernigh t. F eEV s fr om 3x10 6 seeded 4T1BR5-L2G cells w er e c oll ected as abov e. F eEV s or 20 µ g S ynomag-D SPIO (f ar-r ed fluor escence ) w er e r esuspend ed in PKH26 membr ane dy e (Sigma-Aldrich, ca t #MINI 26), as p er manuf actu r er s sug ges tion . F eEV s and SPIO w er e w ashed twice with PBS p rior t o adding to 4T1BR5-L2G cells. Cells an d F eEV s or SPIO w e re incubat ed a t 37°C and 5% C O 2 f or 24-h. Cells w er e t hen w ash ed and fix ed with 4% par af ormaldeh y de (PF A). The chamber w as r emov ed and c ells w ere c ov e r slipped using Flu or omou n t-G mou n ti ng medium with D API (In vitrog en , ca t #00-4959-52). Sections were imaged using a Leica DMi 8 Thunder microsc op e equipped wit h a DF C9000 GT C sCMOS c amer a and LA S-X softw ar e (Leic a , W etzlar , Germ an y). Lar g e v olume c ompu t ation al clearing (L V CC) w as perf ormed on the images. Ima g es w er e p r ep ared using Fiji softw ar e 102 . Nan opar ticle T ra ckin g An alysis F eEV particle siz e a nd c once n tration w e r e measu r ed using a Z etaView Nanopa r ticle T r acking Analy z e r (P article Metrix , Ge rman y). An a v er a g e of 50-150 particles w as r ead p er fr ame as quality c ontr o l. The analy sis par ame t e r s used were: M ax Area:1000, Mi n A r ea : 10, Min Brightness 22, with 11 fr ames rea d twice per sample . T ransmission Elec tro n Micros c o py EV s and F eEV s w er e visualiz ed via tr ans mission electr on micr oscopy (TEM; JE OL 1400-Flash T r ansmission Electr on Microsc op e, Ja pan Elect r on Op ti cs Labor a tory , Ja pan). F ollowing fix ation i n 16% PF A, F eEV s w er e allow ed t o absorb on 200-mesh, c ar bon- c oat e d F orm v ar c oppe r grids f or 20 min, be f or e fixation in 2.5% EM-gr ade glut ar ald eh y de in 0.1M phosp hat e buf f e r ed saline (PBS) f or 15 min a t r oom t emp er ature. The grids w er e s tained with 2% ur an yl ace tat e f or contr ast and w ash ed with EM-gr a de PBS and HPL C-gr ade w at er b e f or e imaging. .CC-BY-NC 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 2, 2024. ; https://doi.org/10.1101/2024.03.12.584146doi: bioRxiv preprint W es t er n Blo t ti ng Cells w er e ly sed in mRIP A ly sis buf f e r (150 mM sodium chloride, 1.0% T riton X-100, 0.25% sodium deo xy chol at e, 50 mM T ris, pH 7.4) consis ting of p r o t eas e inhibi t o r (Ther moFisher , A32955) an d phosphat as e inhibi t or (ThermoFisher , A32957). The supe rnat a n t was used as cell ly sates. Pr o t ei n c oncentra tion of 4T1BR-L2G cell ly sat e, FeEV s, and EV s isola ted using Ex oQuick (S y s tem Biosciences, CA , USA) w as det ermine d using the Pier ce BCA Pr ot ein Assa y kit (ThermoFisher , 23225) using BSA as a s t and ar d . The pr o t ein qua n tification cur v e w as c ompleted in r epli c ate 3 times, a nd the unknowns w ere all r epli c at e d twice. A t o t al of 15 µg of pr ot ein w as a dded p er w ell, mi x ed with DI H 2 O an d RunBlu e LDS sample buf f er (4X) (Expedeon, NXB31010). The cell ly sa te mixtu r es w ere heated a t 70°C f or 10 min, while the F eEV s and EV s w er e n ot he at ed t o a void ag gr eg ation. T he pr o t eins wer e s eparat ed using Mini-PR O TEAN T GX St ain-F r e e Pr e-c as t g els (Bio Rad, 4568093) a t 100 V f or 80-90 min in the BioRa d Mini-Pr o t ea n T etr a s y stem and tr ans f er r ed t o a nit r ocellul ose membr an e using the BioR ad T r ans-Blot T u rbo T r an s f er S ys tem, running at 25V f or 30 mins. The membr ane w as the n block ed using 5% w/v non-f a t dry milk in TBST f or one hour at r oom tempera tu r e a nd th en incub a ted with primary a n tib ody (an ti-Ali x, 1:5000; Pr ot ein T ech, 12422-1- AP or an t i-Flotillin-1, 1:5000 ; Fisher Scie n tific, BDB610820) a t 4°C ov ernight. Th e membr ane w as the n w ashed th r ee times using TBST , and incubat ed in se c onda ry antibody (An ti-r ab bit HRP-link ed, 1:2000) a t r oom t emperature f or on e ho ur . Th e m embr ane was w ash ed ag ain th r ee times with TBST , b e f or e th e Pier ce E CL W es t e rn Blo t ting Subs trat e k it (ThermoFisher , 32209) w as added. T he pr o t eins and ladd er w er e then ima g ed using th e ChemiDoc MP imaging s y s t em (Bio-Rad Labor at ori es, Inc .) using the aut o e x posure and chemiluminescence to obser v e the bands and 635 nm of li gh t with aut oexposu r e f or visualiz a tio n of the ladd er . L C/MS/MS Analysis Pr ot ein s olutio ns w e r e mi x ed with 100mM T ris-HCl (pH 8.5) supplement ed t o 4% (w/v) sodium deo xy chol at e (SDC) t o 270 µl. Samples w er e reduced and alkyla ted by adding T CE P and chlor oacet amide a t 10mM and 40mM, r espec tiv ely and incuba ting f or 5 min a t 45°C with shaki ng a t 2000 rpm in an Eppendorf Thermo Mix er C. T rypsin, in 50mM ammonium bic arbon at e , w as adde d a t a 1:50 ratio (wt/wt) and th e mix ture w as incub at ed a t 37°C ov ernight with shaking a t 1500 rpm in t he Thermomi x er . Fina l v olume of each dig es t w as ~300 µl. Aft e r dig es ti on, SDC w as r emov ed by phase e x tr actio n. The samples w er e acidified to 1% TF A and subject ed to C18 solid phase clean up using St a g eTi ps1 t o r emov e sal ts. An injection of 5 µl (~600ng) w as aut oma ti c ally made using a Thermo (www . ther mo.c om) E AS Y nL C 1200 ont o a The rmo Acclaim P epMap R SL C 0. 1mm x 20mm C18 tr apping c olumn and w ashed f or ~5 min with buf f er A. Boun d pep tides were t hen elu ted ov er 35 min o nt o a Thermo Acclaim P epMap R SL C 0.075mm x 500mm r eso lving c olumn with a gr adi en t of 5%B t o 40%B in 24 min, ramping t o 90%B at 25 min a nd held at 90%B f or th e dura tion of the r un (Buf f er A = 99.9% W at er/0.1% Form ic Acid, Buf f e r B = 80% Acet oni tril e/0.1% F ormic Acid/19.9% W a t e r) a t a c ons t a n t flow r a te of 300 nl/mi n. Column t emper ature w as main tained a t a const a n t t emperature of 50°C using and in t egr at e d c olumn oven (PR SO-V2, Sona ti on GmbH, Biberach, G erman y). Elut ed pepti des w ere spr a y ed in to a ThermoScien tific Q-Ex activ e HF-X mass spectr ometer (www . thermo.com) using a Fle xSpr a y sp r a y ion sour ce. Sur v ey sc ans were tak en in the O rbi trap (60000 r esolu tion, de t ermin ed at m/z 200) an d the t op 15 ions in each sur v ey sc an ar e then subjec t ed t o .CC-BY-NC 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 2, 2024. ; https://doi.org/10.1101/2024.03.12.584146doi: bioRxiv preprint aut om a tic high er en er gy c ollisi on induc ed dissociation (HCD) with fr agmen t spectr a acqui r ed at 15000 r esolu tion. Da t a Analy sis w as perf ormed as f ollow s. The r esulting MS/MS spectr a w ere c on v e rted t o peak lis ts using Ma x Quant2, v1.6.3 .4 (www .ma x qua n t. or g), and sear che d ag ains t a pro t ein dat abase c ont aini ng all mouse sequ ences a v ail able from Uni pr ot (download ed fr om www . unipr o t. or g , download ed o n 20221114) and appended with c omm on labor at o ry c ont amina n ts using the Andr omeda3 sear c h algorithm, a par t of the MaxQua n t e n vir onment. The M a xQua n t ou tpu t w as then analy z ed usin g Sc af f old, v5.1.2 (www .pr o t eomes oftw a r e. c om) t o p r obabili s tically v alidat e p r otein ide n tifications . Assignmen ts v alid at ed using th e Sc af f old 1% FDR c onfidence filt er a r e c onsid ered t rue. Super R esolu tio n Micros c op y Isolat e d F eEV s fr om 4T1BR5-L2G cells w er e ana ly z ed using the ON I EV Pr ofiler ki t, a phosphatidylserin e- based c ap ture reagent, appli ed t o t he EV chip c aptu r e surf ace . The EV sample w as then a pplied a nd fix ed t o th e surf ace with ONI EV fix a ti on buf f er . Label ed antibodies ag ainst t e tr aspa ni ns (C D81-CF647, CD63 - CF568 and CD9- CF488A) w er e appli ed to the c aptu r ed EV s, f ollow ed by ano the r fix ation st ep . dST OR M imaging buf f er w as applied t o the samples and imag ed on the ONI N anoimager using dST ORM imaging c onditio ns: 30°C, 52 ° illumina tion angle (TIRF), 30 m s e xposur e per fr ame. The f ollowing laser s w er e used, seque n tially , in a 3000-fr ame ligh t pr ogr am: 1000 fr ames of each 640, 561 and 488 laser s. Analy sis w as perf ormed using ON I’ s cloud-bas ed pla tf orm, CODI. In Vivo Studi es Six-w eek old f emale Balb/C mice w er e pur chased fr om Charles Riv er Labor at o rie s, and k ept in the MSU animal f acilities with appr ov al fr om the MSU Ins ti tutio nal Animal Car e and Use Commit t e e.@iO;Mic e which did not h a v e t umor s receiv e d F eEV s (fr o m 4T1BGL; n=2, 1 .45x10 10 ) or SPIO (S yno mag-D f ar r ed, n=2, 8 .2 μg). F ollowing i. v . adminis tr a ti on, t he mic e w ere imag e d using MPI and CT (described below) a t 24-h, 48- h, and 7 d pos t injecti on to obser v e th e l oc aliza tion of ir on. Primary tumor s were est a blished by inj ecting 3x10 5 4T1L2 cells int o th e mam mary f a t p ad (MFP) and e xpe rime n ts wer e initi at ed 3-week s af t e r injection . A sampl e of FeEV s w ere ima g ed using MPI p rior to injection to determine a n es tim at e of ir o n pr ese n t . 4T1L2-deriv ed F eEV s (n=5, 1.7 x10 10 ) or equal amount SPIO (S ynomag-D , n=3, 9.6 μg ) w er e inject ed int o t he tumor (intr atumor al ; i. t.) in 25 μl PBS. F ollowing i. t. adminis tra tio n, mice w ere imaged with t he st anda r d 2D imaging mod e in MPI an d CT (described b elow). F ollowing the final imaging time p oint, mice w ere sacrificed using 5% carbon d io xide, and und er w ent pos t-mor t em dissec tion t o r emove tumo r s. Experime nt al br ain me t a s t asis were initi at ed in mice via i n tr a c ardiac (i.c.) injecti on of 2x10 4 4T1BR5-L2G cells, r esusp end ed in 85 µl PBS mi x ed w ith 15 µl ultrasound micr ob ubbles (FU J IFI LM VisualSonics, W A, USA). Mice w e r e anesthe tiz ed (2% isofluor ane in o xy gen), f ollow ed by applicat ion of a de pilat o ry t o r emov e fur on th eir chest. Subcu t an e ous adminis tra tio n of k et oprof en (5 mg /kg) w as used as an analg esic. The mice w ere placed supine, with their ext r emiti es secur ed, and t he le ft v entricle of th e hear t w as loc a ted f ollow ed by guidance of the needle and injection of the cell/micr obubble mixtu r e using ultr asound (V ev o 2100, Visualso nics). Mice w ere monitored f or br ain met ast asis (r el a ti ng t o luminescence) using the IVIS Spectr um ( P erkinElmer). One hundr ed micr oliter s of D-Lucif erin (P erkinElmer , CT , USA, c a t #122799; 30 mg /ml) w as inject ed i.p. 15 min prio r to imaging , immedia tely .CC-BY-NC 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 2, 2024. ; https://doi.org/10.1101/2024.03.12.584146doi: bioRxiv preprint f ollowing i.c. cell injection and ev e ry 4 8-h un til FeEV adminis tra ti on. A sample of F eEV s w ere imaged using MPI prior t o injecti on t o de t e r mine an es tim a te of ir on pr es ent. 4T 1BR5-L2G-deriv ed FeEV s (6.97x10 10 ) or SPIO (30 or 36 µg) w er e inject ed i.c . 7-8 da y s f ollowing br ain me t ast asis est abl ishment . Mice w e r e giv en analgesic (k et oprof en , 5 mg /kg) subcut aneous prior to beg inning the p r ocedu r e . Ultr asound w as used (as abov e) t o admi nis ter F eEV s or SPIOs. F ollowing i.c. injec tion, mice w ere imag ed using MPI and CT (described below). In Vivo Im agin g Imaging w as perf ormed a t th e time- poin ts descri bed, using the f ollowing par amet e r s. MPI w as perf ormed using the st anda r d 3D and 2D imaging modes (F O V 12×6×6 cm, 5.7 T / m gr adien t, 1 (2D) or 21 (3D) pr ojections, and 1 a v erag e). Mic e w e r e the n trans f erred t o a Qu antu m GX micr oCT sc an ner (P erkinElmer). Whole-body CT imag es wer e acquired using 3×8 s s c ans with the following par ameter s: 90 kV v olt ag e , 88 μA amperag e , 72 mm acq uisition FO V , a nd 60 mm r e c onstructi on FO V , resulting in 240 μm v o x els S t and ar ds of known ir on amou n t w er e pl aced in th e MPI bed t o ai d in c o- r egis tration of μCT and MPI sc ans. Ima ge An alysis MPI dat a sets were visualiz ed and a nal y z ed using Horos imaging softw a r e (Hor os is a f r ee and ope n sour ce c od e softw a r e pr ogr am that is dis tribu t ed free of char ge unde r the L GPL license at Hor ospr ojec t.o r g and sponso r ed by Nimb le Co LL C d/b / a Purview in Annapolis, MD , USA). MPI quantific ation w as pe rf ormed on 2 D imag es. Signal threshold w as chose n by selecting an ar e a of backgr ound fr om the imag e which did not ha v e an y ir on present (primary tumor : adjacen t gut signal and br ain: signal outsid e of the head). 3x sd ev of the backgr ound was set as a low e r th r eshold t o captu re signal abov e this v alu e using th r esholdi n g in Hor os. F o r prima ry tumor q uantification, the mea n signal of the gut (minus the nois e fr om a blank i mag e) w as subtr acted fr om the me an si gnal fr om the tumo r t o acc ount f or an y signal which w as added t o the tumor . In ins tances where the si gnal w as low ( i.e ., SPIO injection, 72-h), if the thresholding spread to include ot her r egions ( i.e . , the ir on fiducials), it w as r emov ed manu ally . Iron signal in F eEV pellets, t he prima ry tumor , o r th e br ain o f mice aft er injection o f F eEV , EV or SPIO w er e det e rmined as abov e, with t otal MPI signal c alcula t e d by mean signal x ar ea. Mass of ir on in the F eEV sampl es and m easure men ts from NT A (abov e) w e r e used t o de t e rmine the amount of ir on per FeEV . Ir on amou n t w as de t ermin ed using c ali br a ti on lines. Dif f e r e n t amou n ts of ir on w er e ima g ed with MPI using imaging sequences t o est ablish a r e f erence cur v e f or each scan type u tili z ed: s tanda r d and high sensitivity 2D or s tanda r d 3D . A simple li near r eg r ession was pe rf ormed t o find t he slope of t he d at a (m) using bes t-fit v alues (y=known ir on c on tent and x = MPI signal) with the x,y in ter cep t set to 0. Th e equation y=mx allow e d f or quantific atio n of the ir on cont e n t in FeEV pellets an d in vivo by subs titut ing the t o t al MPI signal (x) fr om the ROI int o the equ ation t o solv e f or ir on (y). Ir on concentra tion v alu es displa y ed on sc ale bar s were de t ermin ed by plotti ng mean signal (x) and ir on/mm 2 or ir on/mm 3 (y , based on 2D or 3D dat a se ts) fr om the i r on amou n ts used f or th e c alibratio n lines. Concentra tion of ir o n w as solv ed f or by input ting mean signal (x) and solving f or y . His t ol ogy .CC-BY-NC 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 2, 2024. ; https://doi.org/10.1101/2024.03.12.584146doi: bioRxiv preprint Br ains and isolat e d primary tumo r s w er e fi x ed ov er nigh t in 4% par af ormaldeh y de f ollow ed by cry opreser v ation th r ough seri al submer s ion in 10%, 20%, and 30% sucr ose f or 24 -h each. Samples w e re then plac ed in a bed of OCT , b e f or e bei ng flash fr o z en in a mixtu r e of dry ice a nd ethanol . The fr o z e n samples w e r e s tored a t -20°C in preparation f or tissue sec tioning using a cry o s tat (Leic a CM3050 S, 10 µm thickness). F or primary tumor s, tissu e sections with f ar-r ed e xpression fr om the ir on nanop articles, as de t ermin ed by scr eening , w ere w ashed with PBS f or 5 min, be f or e b eing added t o 0 .3% tr it on X-100 in PBS and incubat ed f or 45 min. Slides w er e then incubat ed in blocking buf f er (5% goa t serum and 0.3% trit on X- 100 in PBS) f or 60 min. Anti-CD47-PE (3 µg /ml; Bioleg end, ca t #127507) and F4/80 Monoclonal Antibod y (1:200; ThermoFisher , ca t #14-480185) w er e th en add ed t o the slid es ov ernig h t a t 4°C f ollow ed by w ashing with PBS. Sections w e r e th en i ncubat ed with a se c onda ry Go a t a n ti-R a t Ig G antibody , AF647 (1:500, ThermoFisher , c at#A-21247) f or 2 hour s a t r oom t emper ature f ollow ed by w ashing with PBS. Sections were imaged using a Leic a DM i8 Thunder microsc op e equi pped wi th a DF C9000 GT C sCMOS c amer a and LAS-X softwar e (Leic a, W etz lar , G erman y). Lar ge v olum e c ompu ta ti o nal clea ring (L V CC ) w as perf ormed on t he imag es . Images w e r e p r epa r ed using Fiji softw are 102 . Br ain secti ons w e r e w ash ed with PBS f or 5 min f ollow ed by P erls’ Prussian Blue (PPB) s t aining to visualiz e ir on. D API moun ting media (Fluor omo un t-G , In vitr ogen) w as used t o visualiz e nuclei. Sections w ere imag ed using a Nik on E clipse Ci micr osco pe equipp ed with a Nik on DS-Fi3 high-definition c am er a (Nik on Ins tr uments Inc. T oky o , Japan) f or c olor and brigh tfield acquisi tion, CoolS NAP D Y NO (Phot omet rics, AZ, USA) f or fluor escen t imaging and NIS ele men ts BR 5.21.02 softw are (Nik on). Imag es w er e pr e pared using Fiji softw ar e 102 . PPB s t aining (blue) w as pseudoc ol or e d mag e n ta f or ov erla y with D API. St atistical A nal ysis St atistic al an aly ses w e r e perf orm ed using Prism so ftw ar e (10.1.1, GraphP ad Inc. , CA, USA). A tw o-w a y r epe at ed measu r es A NO V A with un c or r ec t ed Fish er ’ s LSD w as use d t o c om par e dif f e r ences in EV associat ed pr o t ei ns betw e en F eEV s and EV s deriv ed fr om 4T1 L2 or 4T 1BR5-L2G cells. A tw o-w a y r epe at ed measu r es AN O V A with unc o r r ec t ed Fisher ’ s LSD w as used t o c ompar e dif f e r enc es in ir on quan tification in primary tumor s betwee n those with that r eceived F eEV s or SPIO , and betw een 0, 24, 48 and 72-h. Dat a are expressed as m ean +/- s t and ar d devia ti on; p <0 .05 w as considered a signific a nt finding. Suppor ting Inf or ma tion R epresenta tive F eEV char act e ris tics and ir on c ont e n t f or F eEV s inject e d in vivo, MPI of S ynomag-D SPIO and F eEV pellet, w e s tern blo t analy sis of F eEV , EV and cell ly sa t e f or 4T1BR5-L2G, super r esolu tio n micr osc opy of F eEV s, in vivo biodistribu ti on of SPIO or F eEV s int o h ealth y mice. A c k n o w l e dg e m en t s W e w ould lik e to th ank Dr s. Micha el Bac hmann, B ry an Smith and P aula F oster f o r pr oviding us with th e cells used. W e acknowledge th e MSU Mass Spectrometry and M etabolomics Cor e f or perf orming th e e xpe rime n ts and pr oviding the protoc ol that w as used f or the analy sis . W e thank the MSU IQ Adv ance d Molecular Imaging F acility f or their help and guidance during a nimal imaging. W e acknowledg e the MSU Cen ter f or Adv anc ed Micr os c opy f or the use of their pr o t ocols, equi pment, and f acilities. W e thank Dr . K anada f or sharing his e xper tise with EV s. W e thank the ap plica tions t eam a t O N I f or their suppor t with .CC-BY-NC 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 2, 2024. ; https://doi.org/10.1101/2024.03.12.584146doi: bioRxiv preprint the sup er resolu tion micr oscopy . This s tudy w as funded by MET A viv or ( A VM ) and the James an d K a thle en Cornelius Endowme n t (CHC). T OC gr aphic w as made with BioR end er . R e f e r enc es 1. F erla y , J. et al . Glo bal Ca ncer Obser va t or y : Cancer T od ay . (2024). 2. Sun, H., X u, J ., Dai, S ., Ma, Y . & Sun, T . 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