1
Ph 1 Radi INTR Tumor detecti orthoto variant utility MATE Determ isother phytate Biodis Spragu perform Elucid Evalua RAW2 were w Inocul penicil inocula diamet MRI: Germa ms, wi calcula admini = 30×3 RESU Determ saturat data su Biodis biodist h in th (PB) s macrop Elucid contras specifi Evalua shown i.e., 16 Detect hypo-i and pre Doxor has hig were fu DISC Phytate chemic physio therefo direct d and tum REFE [1] Ste Hill R Harisin ACKN This w hytate-Comp iology, Seoul Nat RODUCTION r-associated macrop ion of tumors with opic murine model t by combining the of this novel antitum ERIALS AND M mination of the Th rmal titration microc e solution. To minim stribution of ICPC ue–Dawley (SD) rat med to confirm the dation of ICPC Upt ation of ICPC as 264.7 macrophages washed with PBS, an lation of B16F1 Me llin-streptomycin. B ated 8-week-old C5 ter . MRI scans were MRI studies were any). For the estimat ith a step size of 1.0 ated using Matlab istration of ICPC (n 30 mm 2 ; matrix size ULTS mination of the Th tion in accordance w upport the use of IC stribution of ICPC tribution and retenti e liver and at 8 h in staining (data not s phages, in particula dation of ICPC Up st, residential perito ic. These data strong ation of ICPC as a in Fig. 2d. The ICP 6-fold higher than th tion of Metastatic ntense LN metastas edominantly engulf rubicin-ICPC Com gh affinity for doxo urther confirmed by CUSSION e has long been use cal nature of phytate ological conditions [ ore distinct from tha detection of tumor m mor-specific drug d RENCES eidl C, NEJM 362, 8 P , Science 224, 998 nghani MG, NEJM NOWLEDGEMEN work was supported plex as a Nove tional University phages (TAMs) accu high specificity. He of metastatic melan ICPC with an anti mor MR agent that METHODS hermodynamics of calorimeter (Microc mize the ability of F C and Identification ts. The biodistributi identity of the cells take by Macropha an Effective Mac were cultured in 10 nd ICPC-labeled ce elanoma Mice: B1 B16F1 cells were tr 57BL/6 male mice e then performed for performed on a 9. tion of relaxation ra 0 ms. Other imagin (MathWorks Inc., n=9). A GE and a sp e = 384×384; 15 slic hermodynamics of with a two-site set m PC as an injectable C and Identificati ion time in C57BL/ n the spleen. Other t hown). These resu ar hepatic Kupffer ce ptake by Macropha oneal macrophages gly suggest that ICP an Effective Macr PC alone had an est hat of ICPC alone (F Lymph Nodes in B sis is clearly detect fed by F4/80 + macro mplex (Dox@ICPC orubicin (22.7 nM). y PB staining. ed for the detection e is ideally suited fo [3]. Activated macro at of iron-oxide nan metastases. Our pre delivery simultaneou 875 (2010). [2] Dald 8 (1984). [6] Sewa 348, 2491 (2003). TS by Korean Ministry el MRI Agent Hospital, Seoul, umulate in various ere, we developed a noma. Beyond the icancer drug. Using achieves both TAM Fe 3+ Binding to Ph cal, USA) [3]. Base Fe 3+ -phytate to chela n of the Cells that ion and retention tim s that absorbed ICPC ages: we used thiogl crophage-specific M 0-cm 2 plates at a de ells were prepared in 6F1 melanoma cell ansfected with an F with 4×10 5 B16F1 r metastatic lymph n .4 T MR scanner w ate (R2*) and relaxi g parameters were: USA). For in vivo pin echo sequence ( ces (0.5 mm); NEX f ICPC: Fig. 1a illu model, in which Fe 3 MRI contrast agent on of the ICPC-A /6 mice are shown i tissues, including th lts support the app ells (Fig. 1e) and sin ages: LPS-stimulat did not ingest ICPC PC is selectively eng rophage-specific M timated r 2 * of 82.9 ± Fig. 2e). B16F1-GFP Melan ed. These findings ophages within tumo ): Fig.3c illustrates In Fig. 3d, fluores of sentinel LNs in or simultaneously b ophages are then ab noparticles, which ca eliminary data also s usly . drup-Link HE, Clin atkar AB, Nucl Med y of Educational Sci t for Tumor A Hyeonjin Seoul, Korea, Re cancers and promo an iron-calcium-phy capability of curren g a Doxorubicin-ICP M-specific MR imag hytate and Prepar ed on our ITC result ate Ca 2+ from the bl Absorbed ICPC: me of ICPC were e C. lycollate-elicited pe MRI Probe: ICPC ensity of 2 × 10 6 ce n PCR tubes. s were cultured in D FG12 lentiviral vect or B16F1-GFP me node (LN) imaging with a transmit-only ivity (r2*) of ICPC TR = 8000 ms; flip o LN imaging in a SE) were used (resp = 8). ustrates the thermod 3+ ions bind one set t, due to its stability Absorbing Cells : n Figs. 1c and 1d, r he brain, heart, lung, plication of ICPC a nusoidal lining cells ed TEPM more eff C. As an additional gulfed by activated MRI Probe: Fig. 2c ± 3.1 mM -1 s -1 . Incub noma Mice In Vivo were confirmed by or regions, as the an the thermodynami scence analysis clea cancer patients in th inding Fe 3+ and Ca 2 ble to engulf these in an also localize to h strongly support tha Cancer Res, 17, 56 d (Stuttg) 14, 46 (1 ience and Technolo Associated M Delivery Kim 1 , and Byung epublic of, 2 Lee G Republic of ote tumor angiogen ytate complex (ICP nt TAM imaging m PC complex (Dox@ ging and tumor-spec ration of ICPC: we ts, a 10 mM solutio lood, equimolar con The clearance of IC examined in C57BL eritoneal macrophag C alone and ICPC-l ells mL -1 in a mediu DMEM complete m tor that expressed g elanoma cells per m g. Regional LNs (lef y volume coil for in vitro, a gradient p angle (FA) = 90º; animals, we perfor piratory-gated and f dynamics of ICPC t of binding sites wi y under physiologic The clearance of respectively. ICPC c , kidney, lymph nod as an MRI agent fo s of the spleen (Fig. ficiently engulfed IC l control, B16F1 me macrophages. c shows the MR im bation of macrophag o: Fig. 3a shows th y GFP fluorescence ntibody staining com ics of Dox@ICPC o arly demonstrated th the form of techneti 2+ ions and even an nsoluble ICPC parti healthy LNs [9]. In s at the novel antitum 695 (2011). [3] Kim 1975). [7] Ege GN, ogy (2010-0024500, Macrophage (T g-Chul Oh 2 Gil Ya Cancer and nesis and progressio PC) as an MRI agen methods, which is lim @ICPC), we also p cific drug delivery si e performed isotherm on of Fe 3+ -phytate w ncentrations of Ca 2+ CPC from blood wa L/6 mice. Immunohi ges (TEPM) from w labeled RAW264.7 um (ICPC concentr medium (Sigma-Aldr green fluorescence p mouse via the left fr ft axillary and brach excitation and a ph echo sequence (GE ; FOV = 60×60 mm rmed MRI scans b fat-suppressed; TR/ obtained by ITC an ith high affinity (4.6 al conditions [3]. ICPC from blood clearance from the b de, and thymus, sho or use in vivo. The . 1f) [6]. CPC in a dose-depe elanoma cells did n mages used for the e ges with various con he pre- and post-co and PB staining (F mpletely overlapped obtained by ITC an hat Dox@ICPC wa ium-phytate (Tc-phy anticancer drug suc icles [8]. The ability summary, we have mor MR agent, Dox@ m OH, Biochemistry , J Nucl Med 19, 1 , 2011-0002622). TAM)-specifi d Diabetes Institu on [1], and thus mi nt that specifically t mited to visualizati resent our prelimin imultaneously. mal titration calorim was prepared by mix ions were added to as investigated after istochemical (IHC) wild-type mice, whic macrophages were ations ranging 0 - 0 rich, USA) with 10% protein (GFP) unde ront footpad [5]. Th hial) and non-region hased array 4-chann ) was used with 8 d m 2 ; matrix size = 25 efore and 4, 8, 16 TE = 335/6.7 ms fo nalyses. Titration of 62 μM) and bind an after i.v. injection blood was rapid. M wed very low level IHC results clearl endent manner, as c ot take up ICPC (F estimation of R2* o ncentrations of ICP ntrast MR images o Fig. 3b). IHC analys d the PB staining (F nalysis. Titration of as selectively targete ytate), an establishe ch as doxorubicin, s y of ICPC to target a developed a tumor- @ICPC, may potent y 49, 10216 (2010). 1362 (1978). [8] Gu ic Image Con ute, Gachon Univ ight be ideal imagin targets TAMs, and ion of TAMs [2], w nary results that stro metry (ITC) experim xing equimolar conc o the Fe 3+ -phytate so r i.v. injection of th ) staining using an a ch represent inflamm e imaged for relax 0.3 mM). After incu % heat-inactivated F er the control of the he tumor was allow nal LNs were harves nnel surface coil for different echo times 56×256; 1 slice (0.5 6, and 24 h after i or GE and 4000/58 m f Fe 3+ into phytate nother set with low n (10 μmol⋅kg -1 ) in Maximal ICPC uptak ls of ICPC uptake, a ly showed that ICP compared to unstim Fig. 2b), confirming of ICPC in vitro. T PC resulted in an r 2 * of the B16F1-GFP sis demonstrated th Fig. 3b). doxorubicin into p ted in tumor regions ed radiopharmaceut subsequently formin activated macropha -specific MRI agent tially achieve both [4] Kuziel WA,2. P udewicz PW, J Ce ntrast and Dr versity, Incheon, K ng targets for non-i validated its efficac we also developed a ongly support the p ments using a Micro centrations of Fe 3+ i olution (pH = 6.0). e agent (10 μmol⋅k antibody against F4 mation-activated cel xation rate (R2*) m ubation for 12 h, th FBS (Gibco, USA) e ubiquitin C promo wed to grow to ~ 1. sted from the mice. r signal reception ( (TEs) ranging 3.14 mm); NEX = 4. R2 ntravenous (10 μm ms for SE), FA = 90 exhibited Fe 3+ bind affinity (31.25 μM) nto SD rats as wel ke in the mice occur as verified by Pruss PC was engulfed b mulated TEPM (Fig g that ICPC is macro The resulting R2* m of 1056.1 ± 105.9 m melanoma mice wh hat ICPC was prefer hytate showed that s of metastatic LNs ical agent for PET ng insoluble particle ages within tumor re t for use in the accur TAM-specific MR i PNAS 94, 12053 (19 ll Biol 87, 427 (19 rug Korea, invasive cy in an an ICPC potential ocal 200 ions and g -1 ) into 4/80 was lls [4]. mapping. he plates and 1% oter. We 0 cm in (Bruker, 4 - 10.14 2* were mol⋅kg -1 ) 0º; FOV ding site ). These ll as its rred at 4 ian blue y tissue . 2a). In ophage- maps are mM -1 s -1 , here the rentially phytate s, which [7]. The es under egions is rate and imaging 997). [5] 980). [9] 1637 Proc. Intl. Soc. Mag. Reson. Med. 20 (2012)
