OPEN ACCESS
`
`
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`molecules
`
`Molecules 2015, 20, 2081-2099; doi:10.3390/molecules20022081
`
`ISSN 1420-3049
`www.mdpi.com/journal/molecules
`
`Article
`Evaluation of the Radiolabeled Boronic Acid-Based FAP
`Inhibitor MIP-1232 for Atherosclerotic Plaque Imaging
`
`Romana Meletta 1, Adrienne Müller Herde 1, Aristeidis Chiotellis 1, Malsor Isa 1, Zoran Rancic 2,
`Nicole Borel 3, Simon M. Ametamey 1, Stefanie D. Krämer 1,* and Roger Schibli 1,4
`
`1 Department of Chemistry and Applied Bioscience of ETH Zurich, Center for Radiopharmaceutical
`Sciences ETH-PSI-USZ, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland;
`E-Mails: romana.meletta@pharma.ethz.ch (R.M.); adrienne.herde@pharma.ethz.ch (A.M.H.);
`aristeidis.chiotellis@pharma.ethz.ch (A.C.); isam@student.ethz.ch (M.I.);
`simon.ametamey@pharma.ethz.ch (S.M.A.); roger.schibli@pharma.ethz.ch (R.S.)
`2 Division of Cardiovascular Surgery, University Hospital Zurich, Rämistrasse 100, 8091 Zurich,
`Switzerland; E-Mail: zoran.rancic@usz.ch
`3 Institute for Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 268,
`8057 Zurich, Switzerland; E-Mail: n.borel@access.uzh.ch
`4 Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute,
`5232 Villigen PSI, Switzerland
`
`* Author to whom correspondence should be addressed; E-Mail: stefanie.kraemer@pharma.ethz.ch;
`Tel.: +41-44-633-74-03.
`
`Academic Editor: Derek J. McPhee
`
`Received: 4 December 2014 / Accepted: 20 January 2015 / Published: 27 January 2015
`
`
`Abstract: Research towards the non-invasive imaging of atherosclerotic plaques is of high
`clinical priority as early recognition of vulnerable plaques may reduce the incidence of
`cardiovascular events. The fibroblast activation protein alpha (FAP) was recently proposed
`as inflammation-induced protease involved in the process of plaque vulnerability. In this
`study, FAP mRNA and protein levels were investigated by quantitative polymerase chain
`reaction and immunohistochemistry, respectively, in human endarterectomized carotid
`plaques. A published boronic-acid based FAP inhibitor, MIP-1232, was synthetized and
`radiolabeled with iodine-125. The potential of this radiotracer to image plaques was
`evaluated by in vitro autoradiography with human carotid plaques. Specificity was assessed
`with a xenograft with high and one with low FAP level, grown in mice. Target expression
`analyses revealed a moderately higher protein level in atherosclerotic plaques than normal
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`arteries correlating with plaque vulnerability. No difference in expression was determined
`on mRNA level. The radiotracer was successfully produced and accumulated strongly in the
`FAP-positive SK-Mel-187 melanoma xenograft in vitro while accumulation was negligible
`in an NCI-H69 xenograft with low FAP levels. Binding of the tracer to endarterectomized
`tissue was similar in plaques and normal arteries, hampering its use for atherosclerosis imaging.
`
`Keywords: atherosclerosis;
`boronic acid-based inhibitor
`
`fibroblast activation protein; carotid artery plaque;
`
`
`
`1. Introduction
`
`The concept of plaque vulnerability has changed the understanding of the pathogenesis of
`atherosclerosis and has led to novel perspectives for diagnostic and therapeutic interventions. The
`development of diagnostic methods to assess plaque vulnerability is considered an urgent priority in
`clinical and basic research [1]. The assessment of plaque vulnerability in patients at risk for
`cardiovascular disease would allow an adequate pharmacological and/or surgical treatment already in
`the asymptomatic stage and, therefore, reduce atherosclerosis-associated disability and mortality.
`Molecular imaging with suitable tracers has the potential to non-invasively identify molecular processes
`providing functional information about disease progression. In the asymptomatic stage, functional
`imaging may thus provide more specific information on plaque vulnerability than morphology-based
`imaging modalities [2]. Several imaging targets and the respective tracers are under investigation with
`the goal to image plaque vulnerability. The most prominent tracer is [18F]fluorodeoxyglucose, which
`accumulates in cells with high glucose consumption, including activated macrophages. However, the
`unspecific mechanisms of accumulation and the high uptake in myocardium limit its applicability [3].
`Nowadays, plaque progression is regarded as a dynamic and complex process with stabilizing and
`destabilizing components involved. If destabilizing plaque components prevail over stabilizing factors
`an atherosclerotic plaque may eventually rupture leading to often severe or even fatal complications.
`Stabilizing components include an intact and thick fibrous cap that is formed by smooth muscle cells
`(SMCs) embedded in an extracellular matrix rich in collagen. On the contrary, plaque vulnerability is
`related to a thinning of the fibrous cap facilitated by the gradual loss of SMCs and the degradation of the
`collagen-rich fibrous cap [4]. The digestion of the extracellular matrix is caused by proteases in the
`atheromata which include matrix metalloproteinases (MMPs), cathepsins S/K and as recently proposed
`the fibroblast activation protein alpha (FAP, seprase) [5–8]. FAP is a type II membrane-bound serine
`protease belonging to the subfamily dipeptidyl peptidase IV N-terminal (DPP IV, S9B) within the prolyl
`oligopeptidase family (POP, S9) [9–11]. In contrast to other members of the DPP IV subfamily, FAP
`displays endo- besides exopeptidase activity [12]. FAP is capable of cleaving peptide bonds between
`proline and another amino acid [12]. FAP has gelatinase activity and is involved in the further digestion
`of degradation products of type I collagen [13–16]. The endo- and exopeptidase enzymatic activity
`requires homodimerization and glycosylation of the protease [10,14,17].
`FAP was initially identified as a pivotal component of the tumor microenvironment expressed by
`reactive stromal fibroblasts in over 90% of common human epithelial carcinomas and may serve as a
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`therapy target in oncology [18–20]. Furthermore, an association of FAP expression with inflammatory
`processes was described [18] and in line with this finding is emerging data by Brokopp et al. indicating
`an involvement of FAP in the pathogenesis of atherosclerosis [7]. In detail, Brokopp et al. showed that
`FAP is expressed by SMCs in human aortic plaques and confirmed its involvement in type I collagen
`degradation in aortic fibrous caps. Moreover, an association between tumor necrosis factor alpha (TNFα)
`secretion by macrophages with FAP expression in cultured human aortic SMCs and additionally a
`positive correlation of FAP-expressing SMCs with the macrophage burden in human aortic plaques was
`described [7]. The extent of FAP expression at different stages in atherosclerotic plaque progression was
`evaluated and revealed an increased FAP expression in advanced aortic plaques and in thin-cap versus
`thick-cap coronary atheromata by immunohistochemistry and immunofluorescence [7]. These findings
`indicate that FAP expression is related to plaque vulnerability with FAP representing an
`inflammation-induced protease in atherosclerosis. In this respect, FAP could serve as a promising target
`for non-invasive atherosclerotic plaque imaging.
`The goal of this study was to evaluate FAP as a target for atherosclerosis imaging with a small
`molecule. Imaging FAP density requires a FAP-selective ligand with high binding affinity. Several
`research groups have pursued to design small inhibitors with high specificity and selectivity towards
`individual serine proteases in the POP family. To selectively target FAP over other peptidases, its dual
`enzymatic activity as endo- and exopeptidase has to be considered. Identifying inhibitors with high
`selectivity for FAP over other DPPs and the most closely related prolyl endopeptidase PREP is
`challenging due to the 48% amino acid sequence identity of FAP and DPP-4, analogous substrate
`preferences and the ubiquitous expression of many proteases of the POP family [9,11]. Most FAP
`inhibitors share the pyrrolidine-2-boronic acid moiety as a common structural motif. The first boronic
`acid inhibitor reaching phase II clinical trials in the field of cancer treatment was ValboroPro (talabostat,
`PT-100), however due to missing selectivity clinical evaluation was terminated [21–23]. ValboroPro
`displayed IC50 values in the nanomolar range to several prolyl peptidases [24]. The introduction of a
`blocked N terminus in the dipeptidyl boronic acid structure led to novel inhibitors that were evaluated
`regarding binding affinity and selectivity [25–29] with the advantage of impeded intra-molecular
`cyclization reactions mediated by the electrophilic boron and an increased selectivity over DPPs that
`lack endopeptidase activity [30].
`Marquis et al. presented a para-iodine substituted benzamido-glycine-boronoproline analog,
`MIP-1232, with an IC50 of 0.6 nM as determined in an enzyme inhibition assay with human recombinant
`FAP [29]. MIP-1232 was 32-fold more potent in inhibiting FAP than PREP. The corresponding Kd value
`of [123I]MIP-1232 in stably FAP-transfected human embryonic kidney cells (HEK-293) was 30 nM and
`different FAP-positive cell lines showed a markedly reduced enzymatic activity under MIP-1232
`treatment compared to baseline conditions [29,31]. The high binding affinity to FAP and the selectivity
`profile in combination with the possibility to radioiodinate MIP-1232 without altering its structure make
`this compound a promising molecule to assess the potential of FAP as an imaging target for the staging
`of plaque vulnerability and to detect FAP-positive tumors that may respond to FAP-targeted therapy. In
`this study, we investigated FAP expression in human carotid specimens by quantitative polymerase chain
`reaction (qPCR) and immunohistochemistry (IHC). Furthermore, we synthesized MIP-1232 and
`subsequently radiolabeled this compound with iodine-125. Its accumulation in human atherosclerotic
`plaques was evaluated in vitro by autoradiography.
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`A FAP-positive SK-Mel-187 melanoma xenograft and an NCI-H69 xenograft with low FAP levels,
`both grown in mice, were used as controls.
`
`2. Results and Discussion
`
`2.1. Gene Expression Analysis of FAP and SMA in Human Carotid Plaques
`
`Quantitative expression analysis of FAP and alpha smooth muscle cell actin (SMA) by qPCR was
`performed with β-actin as reference gene (Figure 1). For FAP, a similar average gene expression was
`determined in normal arteries, stable plaques and vulnerable plaques (Figure 1A). The average SMA
`gene expression was not significantly different comparing vulnerable and stable plaques (Figure 1B).
`No significant correlation between the SMA and FAP gene expression in human endarterectomized
`plaques was observed (Figure 1C).
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`
`Figure 1. Relative mRNA expression levels of FAP (A) and SMA (B) in normal arteries
`(n = 2), stable plaques (n = 11) and vulnerable plaques (n = 9). For both proteins no
`significant difference was detected between stable and vulnerable plaques. (C) Comparison
`of the relative mRNA expression levels of FAP and SMA. mRNA expression was quantified
`by qPCR, shown are averages of three independent analyses. Lines indicate mean values. The
`square bracket indicates an outlier that was excluded from statistical analyses.
`
`2.2. Immunohistochemical Staining of Human Carotid Plaques for FAP and SMA
`
`The expression of FAP and SMA was further investigated by immunohistochemistry in consecutive
`sections of human atherosclerotic plaques (Figure 2). Normal arteries were FAP negative. In plaques, a
`focal FAP expression in macrophages and giant cells located in the superficial regions of the fibrous cap
`was observed with the most pronounced focal signals in vulnerable plaques (Figures 2C2,D1,D2). SMA
`was strongly expressed in the tunica media in all three classification categories with the highest
`expression in the vasa vasorum of normal arteries (Figure 2A1). The distribution pattern of SMA within
`atherosclerotic plaques was generally focal with major clusters in the cap or shoulder region.
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`A normal artery
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`Figure 2. Hematoxylin/eosin (HE; A–C) and immunohistochemical (A–D) staining for FAP
`and SMA of representative 2 μm paraffin-embedded sections of a normal artery (A), a stable
`plaque (B) and vulnerable plaques (C,D). Boxed higher-magnification images show a small
`blood vessel (normal artery A1), regions in the fibrous cap (stable B1, B2 and vulnerable
`plaque C1) and FAP-positive macrophages (C2, arrows). (D) High magnification images
`show FAP-positive giant cells (D1, arrowheads) and macrophages (D1, D2, arrows) in a
`vulnerable plaque. The endarterectomized plaques are composed of tunica intima and part
`of the media. Lu: lumen. Scale bar, low magnification 2000 μm; A1, B1, B2, C1, 200 μm;
`C2, D1, D2, 50 μm.
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`No distinct co-localization of the two expression markers was found in all examined carotid plaques
`(Figure 2B1,B2).
`
`2.3. Chemistry and Radiochemistry
`
`Reference compound and precursor were synthesized from commercially available 4-iodobenzoic
`acid and glycine ethyl ester hydrochloride, as shown in Scheme 1. The synthetic scheme followed was
`the one reported by Zimmerman et al. [31] with some distinct modifications. For the reference
`compound, glycine ethyl ester was efficiently coupled to 4-iodobenzoic acid with HBTU as the coupling
`agent to afford compound 1 in 79% yield. The ethylester was then cleaved under basic conditions
`(aq. KOH/MeOH) to give the free acid 2 in moderate yield (55%) [32]. Reaction of compound 2 with
`(R)-boroPro-(+)-pinanediol·HCl using the EDC/HOBt coupling system afforded dipeptide 3 in excellent
`yield (93%).
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`Notes: Reagents and conditions: (a) HBTU, DIPEA, DMF, rt, 3 h, 79%; (b) KOH, MeOH/H2O, rt, 1 h, 55%;
`(c) EDC, HOBt, (R)-BoroPro-(+)-pinanediol·HCl, DCM, 0 °C to rt, 16 h, 93%; (d) NH4OAc, NaIO4,
`acetone, rt, 17 h, 45%; (e) hexamethylditin, Pd(PPh3)2Cl2, dioxane, rt, 3 h, 81%; (f) EDC, HOBt,
`(R)-BoroPro-(+)-pinanediol·HCl, DCM, 0 °C to rt, 16 h, 59%.
`
`Scheme 1. Synthesis of reference compound 4 and corresponding precursor 6.
`
`Deprotection of the boronic ester to the free boronic acid, proved challenging. The proposed
`transesterfication method [31] with phenylboronic acid was not efficient in our hands. Apart from
`solubility problems the reaction was sluggish producing many byproducts. Thus, an alternative method
`was applied, which involved oxidative cleavage of the pinanediol protecting group using sodium
`metaperiodate [33]. This procedure was more compatible with our substrate, yielding cleanly and
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`relatively fast a new more polar product as revealed by HPLC monitoring. After workup, the crude was
`purified with preparative RP-HPLC to provide 4 in moderate yield (45%).
`For the synthesis of the precursor, a similar procedure was followed. Stannylation of the iodinated
`compound 2 was achieved by reacting it with hexamethylditin and Pd(PPh3)2Cl2 in refluxing dioxane,
`which yielded compound 5 in a good yield (81%). 5 was then coupled to the boronic ester
`(R)-boroPro-(+)-pinanediol with EDC/HOBt in DCM and the crude was purified with RP-HPLC to
`provide the precursor 6 in satisfactory yield (59%).
`[125I]MIP-1232 was produced in a one-step reaction by electrophilic radioiodination of the
`corresponding trimethylstannyl precursor (Scheme 2). The procedure was performed according to
`Zimmerman et al. [31] with some modifications since the order of addition of the reagents critically
`affected the outcome of the reaction. The experimental protocol was optimized so as to yield a reliable
`and robust radiolabeling procedure. Briefly, precursor 6 was incubated with Na[125I] under oxidative
`conditions to achieve electrophilic radioiodination and simultaneously cleaving the boronic acid
`protecting group. After quenching with Na2S2O3, the reaction mixture was purified by analytical HPLC
`to yield [125I]MIP-1232 in 10%–12% radiochemical yield (decay-corrected; n = 3) and radiochemical
`purity ≥ 90%.
`
`/
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`Note: Reagents and conditions: Na[125I], H2O2, H2SO4, CH3COOH, MeCN, rt, 10 min then Na2S2O3.
`
`Scheme 2. Radioiodination scheme of precursor 6 to [125I]MIP-1232.
`
`The stability in acetonitrile/water/TFA (as eluted from the HPLC column) was investigated by HPLC
`with reference compound 4. The compound was stable with >96% intact compound present after 110 h
`storage. [125I]MIP-1232 was stored under identical conditions and all experiments were performed
`within this time period after purification.
`
`2.4. In Vitro Autoradiography
`
`Radiotracer binding was evaluated by in vitro autoradiography with human carotid plaques and
`xenograft tissue, as shown in Figure 3. [125I]MIP-1232 binding was higher in atherosclerotic plaques
`than normal arteries. Vulnerable plaques showed a slightly higher radioactivity signal integrated over
`the tissue slice than stable plaques. However, after correction for the size of the tissue samples, average
`total binding was similar for the three categories (Figure 3A,B). Radiotracer binding was reduced under
`blockade conditions with an excess of unlabeled MIP-1232 indicating displaceable (specific) binding of
`[125I]MIP-1232 (Figure 3A). No significant difference was detected comparing the specific binding of
`the three groups (Figure 3B). In a proof-of-principle study, target specificity of [125I]MIP-1232 was
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`evaluated in an autoradiography assay with xenograft tissue (Figure 3C). FAP-positive SK-Mel-187
`melanoma xenografts [34] displayed a markedly higher radioactivity signal than NCI-H69 lung small
`cell carcinoma xenografts and radiotracer binding was blocked completely by excess of MIP-1232 in
`both xenografts. IHC experiments confirmed high FAP levels in the SK-Mel-187 xenograft and low
`levels in the NCI-H69 xenograft (Figure 3C).
`
`2088
`
`A
`
`Normal arteries
`
`Stable plaques
`
`Vulnerable plaques
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`Figure 3. (A) In vitro autoradiogram of representative sections of human carotid plaques
`under baseline ([125I]MIP-1232) and blockade condition ([125I]MIP-1232 with excess unlabeled
`MIP-1232). Hematoxylin/eosin (HE) staining below represents plaque morphology. Scale bar
`3 mm. (B) Quantified total and specific binding of [125I]MIP-1232 to normal arteries (n = 5),
`stable plaques (n = 16) and vulnerable plaques (n = 15) determined by autoradiography and
`corrected for tissue size. No significant intergroup differences were determined. Lines
`indicate mean values, diamonds indicate the specimens shown in A. (C) In vitro
`autoradiography with xenografts under baseline and blockade conditions. IHC staining for
`FAP of the SK-Mel-187 and the NCI-H69 xenograft (20 µm cryosections). Scale bar 3 mm
`for autoradiography; 50 µm for IHC images. Color scales for minimal to maximal binding.
`
`2.5. Discussion
`
`Recent studies suggest that inflammation-related processes provide promising targets for the
`non-invasive imaging of plaque vulnerability [35–37]. We lately identified the co-stimulatory molecule
`CD80 involved in T cell activation as a promising imaging target since its expression is increased in
`vulnerable plaques. A radiolabeled specific inhibitor accumulated in human vulnerable plaques in vitro [38].
`Moreover, we evaluated an F-18 labeled folate derivative targeting activated macrophages that
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`accumulated stronger in atherosclerotic plaques than normal arteries [39]. Brokopp et al. presented
`another inflammation-related target, FAP, that displayed increased levels in advanced plaques indicating
`an association with the process of plaque destabilization [7]. The mechanistic role of FAP in
`atherosclerosis remains vague. Collagen, thereof 70% collagen type I, is a primary component of the
`extracellular matrix in atherosclerotic plaques [40]. Synergistically with matrix metalloproteinases, FAP
`is capable of degrading type I collagen and these proteases therefore have a destabilizing effect on
`atherosclerotic plaques [15]. The involvement of serine proteases, in particular of the DPP IV subfamily,
`in atherosclerosis and its clinical adverse events certainly warrants further investigations.
`In this study, we analyzed plaque specimens obtained from the carotid artery that showed similar FAP
`mRNA levels as normal artery segments, independent of plaque vulnerability. In agreement with
`Brokopp et al., FAP protein levels as determined by IHC correlated with plaque progression, with the
`highest focal staining in vulnerable plaques. The discrepancy between mRNA and protein levels could
`indicate lower degradation of FAP in vulnerable than stable plaques, in line with differences in protease
`and protease inhibitor levels in the two lesion types [8]. Overall, the difference in FAP expression
`between normal arteries and plaques was modest in our study. In contrast to the publication of
`Brokopp et al., we found FAP protein in macrophages and giant cells within the plaque and found no
`co-localization with SMCs. However, we want to point out that we evaluated artery segments of a
`different location and used different tissue preparations than Brokopp and colleagues. The localization
`of FAP in macrophages in our study is in agreement with recent reports showing FAP expression in M2
`macrophages [41,42].
`The FAP inhibitor MIP-1232 was successfully synthesized and radiolabeled with iodine-125, a
`long-lived gamma ray-emitting nuclide. The synthesis and radiolabeling were accomplished in reasonable
`yields and purity. In a proof-of-principle study with a FAP-positive SK-Mel-187 xenograft [34], a high
`and displaceable binding of the radiotracer was observed, whereas binding to the NCI-H69 xenograft with
`low FAP levels was negligible. This indicates binding of [125I]MIP-1232 to FAP-positive tissue in vitro.
`The potential of this radiotracer for atherosclerotic plaque imaging was investigated by in vitro
`autoradiography with human carotid plaques. Here, we found a pronounced binding to carotid plaques,
`however with no difference in average specific binding between stable and vulnerable plaques and
`between plaques and normal arteries, after correction for the size of the tissue samples. However, 3 of
`the 16 stable and 4 of the 15 vulnerable plaques showed several-fold higher specific accumulation than
`normal arteries. Only a prospective study would show whether this is of clinical relevance.
`Based on our data we cannot conclude on the selectivity of [125I]MIP-1232 for FAP. In the absence
`of a known selective inhibitor, we investigated specificity by blocking with the unlabeled compound
`itself. The relatively high amount of remaining radiotracer after blocking must, therefore, accumulate
`with low affinity. Lipophilicity is most probably not involved as clogP of MIP-1232 is about 0.5. The
`non-specific accumulation may result from interactions with highly abundant hydrolases or other
`proteins with affinities in the high micromolar range, considering that our blocker concentration was
`100 µM. Specificity analysis of MIP-1232 was performed exclusively with FAP and PREP [29]. A
`conclusive evaluation of the binding affinity to dipeptidyl peptidases such as DPP-2, DPP-4, DPP-8 and
`DPP-9 would be required, irrespective of the fact that DPPs display in general low affinities for N
`blocked peptides [43,44]. As [125I]MIP-1232 did not selectively accumulate in the atherosclerotic tissue
`and as its low FAP/PREP affinity ratio is already known we did not further investigate its selectivity
`
`
`Petitioner GE Healthcare – Ex. 1008, p. 2089
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`

`

`Molecules 2015, 20
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`profile. For future studies more selective inhibitors are needed to reduce non-specific tissue
`accumulation. To overcome limitations in specificity, novel lead structures and the use of antibodies and
`fusion proteins was proposed to minimize off-target effects [24,34,44].
`Our findings with the tumor xenografts are of interest in oncology [34,45,46]. Although FAP as a
`target may be of little relevance for tumor imaging in general considering the high diagnostic value of
`[18F]fluorodeoxyglucose; FAP imaging with a selective ligand would enable the identification of
`FAP-positive tumors sensitive to a FAP-targeted radiotherapy with existing antibodies [34].
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`3. Experimental Section
`
`3.1. Patient Characteristics and Human Carotid Tissue Banking
`
`Human atherosclerotic plaque tissues were excised during carotid endarterectomy (CEA) surgery at
`the University Hospital Zurich using the bifurcation advancement technique [47]. The atherosclerotic
`material was removed from the common, external and internal carotid artery. Before surgery, written
`informed consent was obtained from all patients. A total of 25 patients were included in this study with
`an average age of 73.1 years (73.1 ± 6.6 y) at surgery and 84% of them male. After CEA, the tissue was
`transferred to RNAlater® solution (Sigma, St. Louis, MO, USA) and stored at −80 °C until further use.
`Excised material was classified into the categories “normal artery”, “stable plaque” and “vulnerable
`plaque” based on a macroscopic visual inspection and a histological examination. The histological
`analysis was performed with standard staining methods (e.g., hematoxylin and eosin) and according to the
`classification system of the American Heart Association as previously described [38,48]. Plaques were
`classified as stable if there was a lipid core separated to the blood stream by an intact fibrous cap with a
`representative cap thickness >500 μm and a minimum cap thickness >200 μm [49]. Vulnerable plaques
`were lesions with a large necrotic core, a thin or ruptured fibrous cap, high infiltration of inflammatory
`cells and neovascularization. The microscopic characterization of all plaques used in this study was in
`agreement with the macroscopic evaluation. In total 7 normal arteries, 25 stable plaques and 23
`vulnerable plaques were used for gene expression analysis (normal n = 2, stable n = 11, vulnerable
`n = 9), immunohistochemistry (normal n = 1, stable n = 4, vulnerable n = 4) and autoradiography (normal
`n = 5, stable n = 16, vulnerable n = 15), respectively. Classified normal arteries were redundant segments
`from the A. iliaca or A. thyroidea. Note the limited availability of normal arteries.
`
`3.2. RNA Isolation, Reverse-Transcription and Real-Time Polymerase Chain Reaction
`
`Total RNA was isolated from human atherosclerotic plaque segments according to the protocol of the
`Isol-RNA Lysis reagent (5 PRIME, Gaithersburg, MD, USA) using a TissueLyser bead-mill system
`(Qiagen, Hilden, Germany). cDNA was generated by the QuantiTect Reverse Transcription Kit (Qiagen,
`Hilden, Germany). Used primers were custom-made oligonucleotides from Microsynth (Balgach,
`Switzerland): human actin β (ACTB) (forward 5'-CATGTACGTTGCTATCCAGGC-3', reverse
`5'-CTCCTTAATGTCACGCACGAT-3', NM_001101), human fibroblast activation protein alpha (FAP)
`(forward 5'-TGAA CGAGTATGTTTGCAGTGG-3', reverse 5'-GGTCTTTGGACAATCCCATGT-3',
`NM_004460)
`and
`human
`alpha
`smooth muscle
`cell
`actin
`(SMA)
`(forward
`5'-GCTGGCATCCATGAAACCAC-3', reverse 5'-TGCCCCCTGATAGGACATTG-3', NM_001613).
`
`
`
`Petitioner GE Healthcare – Ex. 1008, p. 2090
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`

`

`Molecules 2015, 20
`
`Quantitative polymerase chain reaction (qPCR) was performed with the DyNAmoTM Flash SYBR®
`Green PCR System (Applied Biosystems, Foster City, CA, USA) using a AB7900 HT Fast Real-Time
`PCR System (Applied Biosystems). Quantification was performed by the 2−∆∆Ct quantification method
`with β-actin as a reference gene [50]. All reactions were conducted in duplicates in three independent
`experiments. Specificity of the amplification products was assured by dissociation analysis. SMA is
`specifically expressed in SMCs of different origin.
`
`2091
`
`3.3. Histology and Immunohistochemistry
`
`Plaques were paraffin-embedded and serial sections of 2 μm were prepared for further histological
`and immunohistochemical investigations. Hematoxylin and eosin (HE) staining was performed
`according to routine procedure to classify plaques into the categories “stable” and “vulnerable”. For
`immunohistochemistry, primary antibodies for FAP (anti-FAP, 1:50, rabbit, polyclonal antibody
`directed against the Fibroblast activation protein, NB100-91763, Novus Biologicals (Littleton, CO,
`USA) and SMA (anti-SMA, 1:400, mouse, monoclonal antibody directed against anti-human alpha smooth
`muscle actin, M0851, Dako, Baar, ZG, Switzerland) were used. Antigen retrieval for the anti-FAP
`antibody was performed using acid buffer (pH 6.0), whereas no antigen retrieval was performed for the
`anti-SMA antibody. The detection system included the OmniUltraMab Kit (Roche, Rotkreuz, ZG,
`Switzerland) for the anti-FAP antibody on the Discovery XT instrument (Roche) and the Dako RealKit
`(Dako) for the anti-SMA antibody on the immunostainer (Dako). FAP IHC staining of a SK-Mel-187
`and a NCI-H69 xenograft was performed with 20 µm frozen sections according to the above specified
`procedure without antigen retrieval. Sections were scanned by a slide scanner (Pannoramic 250, 3D Histech,
`Sysmex, Horgen, Switzerland). HE and IHC staining were analyzed by a pathologist (N.B.).
`
`3.4. Chemicals and Reagents
`
`All reagents and starting materials were purchased from commercial suppliers and used without
`further purification. All solvents used for reactions were obtained as anhydrous grade (puriss., dried over
`molecular sieves, H2O <0.005%) from Acros Organics (Geel, Belgium) and were used without further
`purification unless otherwise stated. Solvents for extractions, column chromatography and thin layer
`chromatography (TLC) were purchased as commercial grade. All non-aqueous reactions were performed
`under an argon atmosphere using flame-dried glassware and standard syringe/septa techniques. In
`general, reactions were magnetically stirred and monitored by TLC performed on Merck (Merck
`Millipore, Schaffhausen, Switzerland) TLC glass sheets (silica gel 60 F254). Spots were visualized with
`UV light (λ = 254 nm) or through staining with anisaldehyde solution or basic aq. KMnO4 solution and
`subsequent heating. Chromatographic purification of products was performed using silica gel 60 for
`preparative column chromatography (particle size 40–63 µm, Fluka, Buchs, Switzerland). Reactions