68Ga-Triacetylfusarinine C and 68Ga-Ferrioxamine E for Aspergillus Infection Imaging: Uptake Specificity in Various Microorganisms

68Ga-Triacetylfusarinine C and 68Ga-Ferrioxamine E for Aspergillus Infection Imaging: Uptake Specificity in Various Microorganisms
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  Mol Imaging Biol (2013)DOI: 10.1007/s11307-013-0654-7 *  The Author(s), 2013. This article is published with open access at RESEARCH ARTICLE 68 Ga-TriacetylfusarinineCand 68 Ga-FerrioxamineE for  Aspergillus  Infection Imaging: UptakeSpecificity in Various Microorganisms Milos Petrik, 1,4 Hubertus Haas, 2 Peter Laverman, 3 Markus Schrettl, 2 Gerben M. Franssen, 3 Michael Blatzer, 2 Clemens Decristoforo 1 1 Clinical Department of Nuclear Medicine, Innsbruck Medical University, Anichstrasse 35, 6020, Innsbruck, Austria 2  Division of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria 3  Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 4  Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic  Abstract Purpose:   68 Ga-triacetylfusarinine C ( 68 Ga-TAFC) and  68 Ga-ferrioxamine E ( 68 Ga-FOXE) showedexcellent targeting properties in  Aspergillus fumigatus   rat infection model. Here, we report on thecomparison of specificity towards different microorganisms and human lung cancer cells (H1299). Procedures:   The  in vitro   uptake of  68 Ga-TAFC and  68 Ga-FOXE was studied in various fungal,bacterial and yeast cultures as well as in H1299 cells. The  in vivo   imaging was studied in fungaland bacterial rat infection and inflammation models. Results:   68 Ga-TAFC and  68 Ga-FOXE showed rapid uptake in  A .  fumigatus   cultures, significantlylower in other fungal species and almost no uptake in other microorganisms and H1299 cells,except for   68 Ga-FOXE in  Staphylococcus aureus  .  68 Ga-TAFC and  68 Ga-FOXE revealed rapiduptake in the lungs of  A .  fumigatus  -infected rats, low accumulation in sterile inflammation and nouptake in bacterial abscess. Conclusions:   We have shown that  68 Ga-FOXE and  68 Ga-TAFC have high uptake in  A . fumigatus   both  in vitro   and  in vivo  .  68 Ga-TAFC showed higher specificity, while  68 Ga-FOXEshowed higher sensitivity. Key words:  Siderophores, Gallium-68, Infection imaging,  Aspergillus fumigatus  , Positronemission tomography Introduction I ron is an essential cofactor for a variety of important cellular processes and, therefore, can be considered as a vital nutrient for virtually all forms of life [1, 2]. Most  microorganisms use special mechanisms to acquire ironincluding production of siderophores [3]. Mainly in the iron- poor environments, microorganisms such as  Aspergillus fumigatus  produce large amounts of siderophores to scav-enge iron (III) and enable its uptake into the organism [4]. It has recently been recognised that iron plays an essential rolein infection in general [5] and in fungal infections in particular. It has been shown that in particular, thesiderophore system is essential for the virulence of   A .  fumigatus  [6].Siderophores are low molecular weight, iron-chelatingligands synthesised by almost all microorganisms for ironacquisition and storage; however, not all aspects of siderophore utilisation by microorganisms are fully under-stood. Luckey  et al  . [7] reported that some bacterial strains Correspondence to:  Milos Petrik;  e-mail:, ClemensDecristoforo;  e-mail:   lost the ability to synthesise siderophores but retained the abilityto utilise siderophores released by other microorganisms.Similarly, Haas [8], Philpott   et al  . [9] and Heymann  et al  . [10] reportedthatseveralfungalspeciesareabletoutilisesiderophores produced by other fungi, indicating the lack of speci 󿬁 city of siderophore systems for particular microorganisms.A great variety of different siderophores are known today,the majority being of hydroxamate, catecholate or   α -hydroxycarboxylate type, each having a high selectivity for iron (III) [11]. The chemistry of iron (III) and gallium (III) isvery similar and was already widely exploited in the  󿬁 eld of nuclear medicine in the use of   67 Ga-citrate.  68 Ga is short-lived,generator-producedisotopethathasrecentlybecomethesubject of great interest for molecular imaging applications using positron emission tomography (PET) [12]. We have recentlyshown thatvarioussiderophores can belabelledwith  68 Ga [13],and  68 Ga-triacetylfusarinine C (TAFC) and  68 Ga-ferrioxamineE (FOXE) are able to detect   A .  fumigatus  infection in a rat infection model using PET imaging [14, 15]. TAFC is a common trihydroxamate-type siderophore of many fungal species (  Aspergillus  sp.,  Fusarium  sp.,  etc .).Although many different forms of fusarinines have beendetected, the cyclic acetylated trimer is regarded as the product with the highest chemical stability [16]. Adjimaniand Emery have even shown that TAFC is able to extract iron from other siderophores and thereby feed the producingmicroorganism with iron from exogenous siderophores [17].Ferrioxamines were srcinally isolated and characterised asferrioxamines A to H. All ferrioxamines are trihydroxamate-type siderophores and are either cyclic or linear. FOXE is a cyclic siderophore with high af  󿬁 nity to iron (III) mainly produced by actinomycetes and other bacteria [18].Here,wereportonthecharacterisationandcomparisonof  invitro  and  in vivo  uptake of   68 Ga-TAFC and  68 Ga-FOXE indifferent microorganisms and human lung cancer cells toevaluate their speci 󿬁 city and sensitivity for   A .  fumigatus infection imaging. Materials and Methods Chemicals All commercially obtained chemicals were of the highest available purity and were used without further puri 󿬁 cation. Siderophoreswere obtained from Genaxxon BioScience GmbH (Ulm, Germany),and  68 Ge/  68 Ga generator, from Eckert & Ziegler Eurotope GmbH(Berlin, Germany).  Radiolabelling  Both siderophores were labelled with  68 Ga using acetate buffer at room temperature (RT) (TAFC) or at 80 °C (FOXE) [13]. Radiochemical purity was determined using reverse-phase high- performance liquid chromatography gradient method and/or instant thin-layer chromatography on silica gel impregnated glass  󿬁  bres, asdescribed previously [13  –  15].  Preparation of Microbial Cultures for In VitroStudies  A .  fumigatus  ATCC46645,  Aspergillus terreus  DSM826,  Aspergil-lus  󿬂  avus  ATCC9643,  Rhizopus oryzae  AS5 and  Fusarium solani AS94 were cultured at 37 °C in liquid  Aspergillus  minimal media (AMM) [19] containing 1 % glucose and 20 mM glutamine ascarbon and nitrogen source, respectively. Iron-containing media were supplemented with 30  μ M FeSO 4 , whereas for iron-limitingcultures, iron was omitted. For all other microbial strains, the iron-replete and iron-limited main cultures were at   󿬁 rst precultured for 18 h and inoculated with a single colony at 37 °C. Such aninoculum was subsequently used for the culturing of the maincultures. The preculture medium for   Candida albicans ATCC90028,  Klebsiella pneumoniae  and  Pseudomonas aeruginosa ATCC9027 was yeast peptone dextrose (YPD) + 0.5 % glucose;that for   Mycobacterium smegmatis  mc 2 155, YPD + 0.5 % Tween80; and that for   Staphylococcus aureus , Roswell Park MemorialInstitute (RPMI) + 1 % casamino acids. With exception of   P  . aeruginosa , the iron-replete main culture medium for all thesestrains was the same as the preculture medium, and for iron-limiting conditions, the ferrous iron chelator dipyridyl was added toa   󿬁 nal concentration of 200  μ M. For   P  .  aeruginosa , the mainculture media were iron-replete and iron-limiting AMM (seeabove), respectively. Iron-de 󿬁 cient conditions were veri 󿬁 ed bydetection of extracellular siderophores production, which isrepressed by iron.  Preparation of Human Lung Cancer Cells for In Vitro Studies H1299 non-small cell human lung cancer cells (ATCC) weremaintained in tissue culture  󿬂 asks (Cellstar, Greiner Bio-One,Kremsmuenster, Austria) in RPMI 1640, supplemented with 10 %( v  /  v  ) heat-inactivated FBS and 1 % ( v  /  v  ) PSG at 37 °C with 5 %CO 2  in a humidi 󿬁 ed atmosphere and grown in monolayer. On theday of the experiment, cells were removed with trypsin-EDTA andused at a density of 1×10 6 cells/ml for the uptake experiments.  In Vitro Uptake of    68 Ga-Siderophores in Various Microbial Media  In vitro  uptake was studied in  A .  fumigatus ,  A .  󿬂  avus ,  A .  terreus ,  C  . albicans ,  R .  oryzae ,  F  .  solani ,  P  .  aeruginosa ,  K  .  pneumoniae ,  S  . aureus  and  M  .  smegmatis  iron-de 󿬁 cient and iron-suf  󿬁 cient cultures.For the monitoring of uptake over time,  68 Ga-siderophores wereincubated in the microbial media for 10, 45 and 90 min at RTwith or without blocking solution (Fe-TAFC or Fe-FOXE) in96-well plates (Millipore, Billerica, MA, USA). The uptake wasinterrupted by  󿬁 ltration of the medium and rapid rinsing withice-cold Tris buffer. The  󿬁 lters were collected and counted in a  γ -counter. M. Petrik et al.: Uptake Specificity of   68 Ga-Siderophores  Siderophore Utilisation Growth Assay To exemplary con 󿬁 rm the ability or inability to take up TAFC, wedeveloped a siderophore utilisation growth assay (Fig. 2). In this agar diffusion assay, the analysed species (10 7 conidia of   A .  fumigatus  or 0.2 ml of   K  .  pneumoniae  preculture, respectively) was poured in 5 mltop agar (iron-limiting AMM medium + 0.7 % agar) on agar plates(iron-limiting AMM medium). Subsequently, 80  μ l of 0.6 mM ligand-free TAFC solution was inoculated into a hole (5 mm diameter) punched into the middle of the plate. The plates were then incubated at 37 °C for 30 h. The ligand-free siderophore diffuses into the growthmedium and chelates the present iron with the highest TAFCconcentrationinthevicinityofthehole.Thegrowthofmicroorganismsunable to take up TAFC-iron is inhibited byhigh TAFC concentrationsin this assay as TAFC iron is here the only iron source present.  In Vitro Uptake of    68 Ga-Siderophores in Human Lung Cancer Cells  In vitro  uptake was studied in human non-small cell lung cancer cellsH1299 type. H1299 cells were seeded at a density of 1×10 6 cells per Eppendorf tube and incubated in triplicates with  68 Ga-TAFC or   68 Ga-FOXE at RT for 90 min. For positive and negative control, Eppendorf tubes containing iron-de 󿬁 cient and iron-suf  󿬁 cient cultures of   A .  fumigatus  were incubated in triplicates with  68 Ga-labelledsiderophores at RT for 90 min in parallel. The incubation wasinterrupted by 2-min centrifugation at 5,000×  g  . The supernatant wascollected and measured in a   γ -counter. Cells sediment was disturbed by 1 ml of glycine and subsequent whirling.  Aspergillus  media werewashedwith1mlofice-coldTrisbuffer.Allsampleswerecentrifugedfor 2 min, and supernatants were measured in  γ -counter. Thereafter,1 ml of 1 M NaOH was added to human lung cancer cells, mixed andcentrifuged for 2 min. The supernatant was again collected andmeasured in a   γ -counter.  Animal Experiments All animal experiments were conducted in accordance with theregulations and guidelines of the Austrian and Dutch animal protection laws and with the approval of the Austrian Ministry of Science (66011/42-II/10b/2009) and Institutional Animal WelfareCommittee of the Radboud University Medical Centre Nijmegen(revised Dutch Act on Animal Experimentation, 1997). Animalstudies were performed using Lewis rats (Charles River Laborato-ries, Wilmington, MA)  In Vivo Imaging   In vivo  uptake was studied in the  A .  fumigatus  rat infection model[15, 16] and in rats with sterile (turpentine oil) and bacterial ( S  . aureus ) intramuscular (i.m.) in 󿬂 ammation. An abscess was inducedin the left calf muscle with approximately 1×10 9 colony-formingunits of   S  .  aureus  in 0.1 ml 50:50 % suspension of autologous blood and normal saline. A sterile in 󿬂 ammation was induced in theright calf muscle by injecting 0.1 ml turpentine oil intramuscularly.During the procedure, animals were anaesthetised. After 24 h, whenswelling of the muscle was apparent, the tracers were injectedintravenously through the tail vein.PET imaging was obtained using an Inveon animal PET/CTscanner (Siemens Preclinical Solutions, Knoxville, TN, USA) [15].Static PET scans of 30 min were recorded at 30 min after i.v.injection of   68 Ga-siderophore or 2-deoxy-2-[ 18 F] 󿬂 uoro- D -glucose( 18 F-FDG), the time established in the previous studies [15]. Results  Radiolabelling and In Vitro Stability Both  68 Ga-TAFC and  68 Ga-FOXE showed high radiochem-ical purity and  in vitro  stability as described in the previousstudies [13  –  15]. Figure 1 displays chemical structures of  studied  68 Ga-siderophores.  In Vitro Uptake of    68 Ga-Siderophores in Various Microbial Media  In vitro  uptake of   68 Ga-TAFC and  68 Ga-FOXE was highlydependent on the mycelia iron load (Tables 1 and 2). Both compounds showed rapid uptake by iron-starved  A . Fig. 1.  Chemical structures of   a 68 Ga-TAFC and  b 68 Ga-FOXE. M. Petrik et al.: Uptake Specificity of   68 Ga-Siderophores   fumigatus  cultures (Table 1), which could be partly blockedwith excess of Fe-siderophore and signi 󿬁 cantly lower uptake by  A .  fumigatus  grown under iron-suf  󿬁 ciency (Table 2).Tables 1 and 2 summarise the uptake values of both  68 Ga-TAFC and  68 Ga-FOXE in various microorganisms andH1299 cells. In bacterial (  P  .  aeruginosa ,  K  .  pneumoniae , S  .  aureus ,  M  .  smegmatis ) or yeast ( C  .  albicans ) cultures, 68 Ga-TAFC revealed virtually no uptake in both iron-de 󿬁 cient and iron-suf  󿬁 cient media.  68 Ga-FOXE showedsimilar results to  68 Ga-TAFC except for   S  .  aureus . In  S  . aureus  iron-de 󿬁 cient media, clear uptake of   68 Ga-FOXE wasobserved. In fungal cultures (  A .  󿬂  avus ,  A .  terreus ,  R .  oryzae ,  F  .  solani ), both  68 Ga-siderophores, especially  68 Ga-FOXE,showed certain uptake in iron-de 󿬁 cient media, which wassubstantially lower in comparison with the uptake in  A .  fumigatus  cultures. Siderophore Utilisation Growth Assay In this assay (Fig. 2),  K  .  pneumoniae  displayed in contrast to  A .  fumigatus  a clear growth inhibition zone, which is in perfect agreement with the  in vitro 68 Ga-TAFC uptake assaythat indicated that   K  .  pneumoniae  lacks TAFC uptake (seeTable 1). As a control, the plates were inoculated with ferricTAFC instead of ligand-free TAFC. In this case, iron tracesthat are not chelated by TAFC are available for growth. Inthis set-up, the growth of   K  .  pneumoniae  was not inhibited,which demonstrates that the growth inhibition is indeed dueto iron chelation by TAFC and not by a potential iron-independent antibacterial activity of TAFC.  In Vitro Uptake of    68 Ga-Siderophores in Human Lung Cancer Cells  No uptake was observed in human lung cancer cells (H1299)for both  68 Ga-siderophores (see Tables 1 and 2). Almost all the radioactivity was found in the supernatant containing cellmedia, and negligible radioactivity was observed in theglycine and NaOH supernatants for both compounds.  In Vivo Imaging   In vivo  PET imaging in the  A .  fumigatus  rat infection modelshowed rapid focal accumulation of   68 Ga-siderophores in thelungs. Whereas no  in vivo  uptake in the lung region wasdetected in non-infected animals, and the only visible organswere the kidneys and bladder (see Fig. 3). ligand free TAFCiron-TAFC  A. fumigatusK. pneumoniae Fig. 2.  Siderophore utilisation assay demonstrating thatgrowth of   K  .  pneumoniae  is in contrast to that of   A .  fumigatus inhibited by ligand-free TAFC. Fig. 3.  In vivo  imaging of the  A .  fumigatus  rat infectionmodel (   left  -  hand side images  ) and non-infected (   right  -  hand  side images  ) animals using  a 68 Ga-TAFC and  b 68 Ga-FOXE,1 h. i.v. postinjection. M. Petrik et al.: Uptake Specificity of   68 Ga-Siderophores
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