Journal of Cancer Research & Therapy NobleResearch Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137 www.nobleresearch.org 0 http://dx.doi.org/10.14312/2052-4994.2013-2 Original Research Open Access

Development of novel approach to diagnostic imaging of lung cancer with 18F-Nifene PET/CT using A/J mice treated with NNK

Galitovskiy V1, Kuruvilla SA2, Sevriokov E2, Corches A2, Pan ML2, Kalantari-Dehaghi M1, Chernyavsky AI1, Mukherjee J2,3 and Grando SA1,3

1

2

3Department of Dermatology, University of California-Irvine, Irvine, CA 92697, USA Preclinical Imaging, Department of Radiological Sciences, University of California-Irvine, Irvine, CA 92697, USA Cancer Center and Research Institute, University of California-Irvine, Irvine, CA 92697, USA Abstract oncology. In this study, we utilized the tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancerDevelopment in A/J miceof novel as anmethods animal of model early fordiagnosis development of lung ofcancer a new is imaging one of the technique major tasks for early of contemporary diagnosis of clinicallung cancer. and experimental Lung cancer

NNK treatment, followed by PET/CT scans with 18F-Nifene that binds to  ofcells lungs in A/J were mice also overexpress obtained ex nicotinic vivo. CT acetylcholine revealed the receptors. presence ofLongitudinal lung nodules CT inscans 8-month were carriedNNK-treated out over mice, a period while ofcontrol 8 months mice after had no tumors. Imaging of live animals prior to necropsy allowed correlation4-made of results nicotinic of tumor receptors load via with PET/CT high affinity.and histopathological PET/CT scans 18F-Nifene was seen in the lungs of NNK-treated mice, whereas lungs of control mice showed only minor uptake of 18 18F-Nifene binding in lung in vivo and ex vivo demonstrated a findings. Significant amount of 4 nicotinic receptor subunits. For comparison,F-Nifene. we performed Quantitative PET/CT analysis studies of with the 18 extentF-FDG, and which amount is used of for the imaging diagnosis of lung cancer. The tumor/nontumor ratioshigher for tumor/nontumor 18F-FDG were lower ratio than due forto selective18F-Nifene. labeling Thus, we of havetumor developed nodules expressinga novel diagnostic abundant imaging approach to early diagnosis of lung cancer using 18F-Nifene PET/CT. This technique allows quantitative assessment of lung tumors in live mice, which is critical for establishing tumor size and location, and also has salient clinical implications.

Keywords: 18F-Nifene; 18F-FDG; PET/CT imaging; nicotinic receptors; NNK; A/J mice; lung cancer

Introduction Acetylcholine (ACh) is an auto/paracrine growth factor for lung cancer [4]. Recent studies have demonstrated Lung cancer is the leading cause of cancer death in industrialized countries with a high mortality rate and Corresponding authors: Grando SA, 134 Sprague Hall, University of 5-year survival rates of 15% [1]. Early detection of lung cancer is essential for early therapeutic interventions [email protected]; and Mukherjee J, B138 Medical California-Irvine, Irvine, CA 92697, USA. Tel.: 949-824-2713; Fax: 949- which can reduce mortality. Minimally invasive methods 824-2993, Email: [email protected] for early detection of lung cancer include computerized Sciences, University of California-Irvine, Irvine, CA 92697, USA. Tel.: Both senior authors contributed equally to this paper. tomography (CT) and positron emission tomography 949-824-2018; Fax: 949-824-2344, Email: (PET). A combination of PET/CT has been shown to have Received 3 March 2013 Revised 4 May 2013 Accepted 20 May 2013; Published a higher sensitivity of detection of the primary tumor and Citation: 29Galitovskiy May 2013 V, Kuruvilla SA, Sevriokov E, Corches A, Pan ML, metastases and also correlates with pathology [2]. The Kalantari-Dehaghi M, Chernyavsky AI, Mukherjee J, Grando SA (2013) 18 18F-FDG) PET/CT is being used Development of novel approach to diagnostic imaging of lung cancer for detection of the primary lung tumor and metastases. with 18F-Nifene PET/CT using A/J Mice treated with NNK. J Cancer Res Ther 1: 128-137. 0 However,F-fluorodeoxyglucose there is still a ( substantial need for improving Copyright: ©2013doi:10.14312/2052-4994.2013-2 Galitovskiy V, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution than is currently possible [3]. This goal can be achieved by License, which permits unrestricted use, distribution and reproduction sensitivity and specificity of PET/CT lung cancer detection in any medium, provided the original author and source are credited. using more specific probes. Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137

129 that the nicotinic class ACh receptors (nAChRs) act as We have developed and validated an imaging probe, central mediators in the cancer signaling pathways, and 18F-Nifene (Figure 1), which is suitable for non-invasive have unveiled previously unknown nicotinergic signaling visualization of 42 nAChRs using PET [30-32]. Normal networks in the lung (reviewed in [5-7]). nAChRs are classic lung distribution and kinetics of 18F-Nifene in mice was representatives of the superfamily of ligand-gated ion characterized by us in a previous work [33]. Binding  and Ca2 and of 18F-Nifene in rat brain correlates very well with the  [8]. The subunit genes encoding a pentameric known distribution of 4 125I- channel proteins mediating the influx of Na 1–10, 1– 2 receptors, as defined by efflux4, ,  of, and K  with  iodoepibatidine [30,18 34, 35] and confirmed in the studies thoughtprotein have to be been limited identified to neurons and designated and skeletal muscles, evaluate utility of F-Nifene as an imaging probe in the 2-knockout mice (36). The goal of18 this study was to contemporary. Although research expression has convincingly of nAChRs demonstrated was originally early diagnosis of lung cancer. Since F-FDG PET/CT is routinely used for diagnosis of human lung tumors [2], comparative PET/CT studies were also carried out with nAChR subunits 2-10 and 2-4 in practically all cell 18 expression of different combinations of the "neuronal" F-FDG. diverse set of proteins, including the nAChRs themselves types [9]. Activation of nAChRs modulates expression of a As an animal model of lung cancer, we chose A/J mice treated with NNK. This model has been employed by us in [10-16], which can contribute to the carcinogenic action a carcinogen and appearance of multiple tumor nodules of tobacco constituents [10, 17-19]. The nAChR subtypes previous studies [37, 38]. The time between exposure to thein lung 3, epithelial4, 5, and cells 7 subunitsare well-characterized. that form nAChR In 1997,channels we tumors display a histological pattern characteristic modulatingfirst demonstrated Ca2 that human bronchial cells express ofin the alveolobronchial lungs is relatively adenomas short: 4 to and 5 months alveolobronchial [39]. The adenocarcinomas [40, 41] resembling the human lung metabolism [20]. These findings have adenocarcinoma that has increased in incidence in the been confirmed in other laboratories [21-23]. United States [42]. Mouse tumors originate from the same and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone cells—the type II alveolar epithelial cells and Clara cells. (NNK)Tobacco-specific (Figure 1), nitrosamines, are nicotine N'-nitrosonornicotine metabolites and powerful lung carcinogens that bind to nAChRs [24, 25]. The lung adenocarcinoma developing in A/J mice exposed to tobacco products is associated with overexpression of nAChRs and relies on nAChR18 signaling [10, 19]. Exposure to nicotine or NNK activates and upregulates Herein, we report the use of F-Nifene in the longitudinal respiratory nAChRs [10, 16, 19, 20, 26, 27]. Bronchial PET/CT studies of lung tumors in A/J mice treated with 4- cell transformation also alters the expression of nAChR  subunit profiles [4, 23, 28]. The nAChR subtypes are targetNNK. Thesefor 18F tumors-Nifene. wereThe results found todemonstrated overexpress that the our overexpressed in a variety of cancers, including lung sensitive methods of early diagnosis of lung cancer may newlymade nAChRs developed providing 18F-Nifene the tumor- PET/CT selective method and specific allows cancer [6, 7, 29]. Hence, development of novel and more be based on visualizing nAChRs on cancer cells. However, quantitative assessment of lung tumors in live mice, which there have been no reports on in vivo visualization of lung is critical for establishing tumor size and location, and has cancer using the nAChR imaging probes. salient clinical implications.

Materials and methods A/J mouse model of lung cancer

from Jackson Laboratories (Bar Harbor, ME). Mice Female strain A/J mice, 6–8 weeks old, were purchased tobacco nitrosamines [43]. The animals were housed in a. Nicotine b. NNN of this strain develop lungad tumorslibitum access when to exposed food and to water, and conventional bedding material. Mice were treatedpolypropylene in accordance boxes with to National Institutes of Health guidelines and as approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Mice were divided into three groups, 12 per group

subcutaneous injection of 100 l in the upper back at a dose(Figure of 1002). Experimental mg/kg once a mice week were for fourtreated weeks. with NNK NNK was by c. NNK d. 18F-Nifene

Figure 1 Chemical structures of 18F-Nifene and NNK. Structure of nicotine, weredissolved injected in DMSO with the in 10xsame concentration solution without and dilutedNNK. The in its metabolites N’-nitrosonornicotine (NNN) and 4-(methylnitrosamino)- corn oil to final concentration. Treatment control mice 1-(3-pyridyl)-1-butanone (NNK), as well as 18F-Nifene. CT/PET control mice were also included because multiple Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137 130

Figure 2 Study time-line. Time-line of CT and PET scans of A/J mice injected with NNK vs. vehicle. CT scans were obtained on all mice at the baseline 18F-Nifene PET scans and 18F-FDG PET scans were acquired on the month 8. and 3, 6, and 10 months after treatment. CT scans might induce lung cancer in A/J mice. Lung tissue scanner (Siemens Medical Solutions, Malvern, PA), which sections from control and NNK-treated A/J mice were obtained following PET/CT scans (after a 24-hr period view, was used for CT acquisitions in combined PET/CT has a resolution of 1.46 mm in the center of the field-of-

polyclonalonce the fluorine-18 antibody to radioactivity 4 nAChR had subunit decayed (Millipore, away) experiments [44]. Abdominal images of the mice were Temecula,and stained CA), with the hematoxylin 4-containing and eosin. nAChR Using subtypes rabbit acquiredobtained withwith thea large detector-source area detector assembly (4096 x rotating 4096 pixels, over 10 cm x 10 cm field-of-view). The CT projections were sections of lung tissue, and also by immunoblotting of of 4 was used in order to increase the signal to noise ratio lungwere homogenates.visualized by immunofluorescence Protein bands were in visualized the cryostat by in360 the degrees images. and The 720 CT rotation images steps. were A reconstructeprojection bind factorusing co

Gilbertsville,affinity purified PA). goat Fluorescent anti-rabbit signal secondary was detected antibody by ne-beam reconstruction with a Shepp filter with cutoff conjugated to IRDye 680 or IRDye800 (Rockland, Inc., studiesat Nyquist were frequency analyzed resulting using Inveon in an Researchimage matrix Workplace of 480 (IRW,x 480 xSiemens 632 and Medical a voxel sizeSolutions, of 0.206 Malvern, mm. Longitudinal PA), software. CT Odysseyscanning 2.1 the software nitrocellulose (LI-COR filter Biotechnology, in a LI-COR Odyssey Lincoln, Volumes of interest (3D VOIs) were drawn manually on the NE).near Visualization infrared imaging of -tubulin platform with and a respective quantified antibody using lungs of control and NNK-treated mice. VOIs were drawn from Millipore was used as a loading control. as irregular contours on the high resolution CT images, 10 months) of the same mice were calculated by using the Radiochemicals segmentationand tumor volumes function at different in the IRW time software. intervals (baseline to Synthesis of 18F-Nifene has been established and was carried out following reported procedures [32]. The 18F-Nifene and 18F-FDG PET imaging : Mice were injected 18 automated radiosynthesis of F-Nifene was carried out with 100 Ci 18F-Nifene or 100 Ci 18F-FDG via tail vein under anesthesia. The animals were then placed in the 2 An Alltech C18 column (10  ) was used mouse chamber and were positioned in the Inveon in the CPCU (chemistry-processing control unit) box. Multimodality CT/PET scanner. The Inveon PET/ of 18F m, 250 x 10 mm CT was switched to the “docked mode” for combined collectedfor reverse-phase fraction was HPLC taken purification. to near dryness Specific in activity vacuo. -Nifene was approximately18F-Nifene 2000 was Ci/mmol. carried The out reconstruction of the PET data and further analysis of thePET/CT PET/CT experiments. data. Duration A CT of scan the PET was scan then was obtained typically for The final formulation of m) into a sterileusing approximatelydose vial for use 2 to in 5the mL PET of 0.9%studies. saline 18F-FDG followed was 18F-Nifene or 18F-FDG. The images were reconstructed 30 min and was timed to start 60 min post injection of purchasedby filtration from through PETNET a membrane solutions filter(Irvine, (0.22 CA). Fluorine- 18 radioactivity was counted in a Capintec CRC-15R dose using fourier rebinning and 2-dimensional filtered back- calibrator, while low level counting was carried out in a projection (2D FBP) method (ramp filter and cutoff at Capintec Caprac-R well-counter. Nyquist frequency) with an image matrix18F of-Nifene 128x128x159, and 18F- FDGresulting were in analyzed a pixel sizeas tumor of 0.77 to mmnontumor and a sliceratios. thickness Similar In vivo CT and PET imaging studies of 0.796 mm. The PET data for both CT Imaging: Prior to imaging, mice were fasted in a dark delineated visually by contouring the 18F-Nifene or 18F- quiet place for  FDGto previously activity thatdescribed was clearly methods above (45), normal the VOIs background were first 6 hours. In preparation for the scans, mice under anesthesia during the scan (2-4% induction and corresponding CT of each animal. The amount of 18F- were anaesthetized with isoflurane and then maintained 2-2.5% maintenance). An Inveon Multimodality (MM) CT Nifeneactivity or in 18 theF-FDG lungs. activity These in areaseach VOIwere (in confirmed kBq/mL) inof the 131 Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137 tumor and areas of the lung that did not have any tumors

CT scans of each individual animal. were measured and confirmed using the corresponding Ex vivo PET/CT studies Mice were decapitated after an 18F-Nifene or an 18F-FDG scan, and the lungs were rapidly removed and frozen. The whole lungs from control mice and NNK-treated mice

(top edge side length 4.5 cm, bottom edge side length 3 cm)were and placed covered in a with hexagonal powdered polystyrene dry ice. weighingThis boat boatwas placed securely on the scanner bed. PET and CT scans were acquired simultaneously of the control and NNK-treated mice lungs for 30 min. Images were analyzed using the Acquisition Sinogram Image Processing (ASIPRO, Siemens Medical Solutions, Malvern, PA) and IRW software from Siemens Medical Solutions.

Results In vivo 18F-Nifene PET/CT studies The longitudinal CT image analysis of the scans showed absence of tumors in control mice and multiple tumors in both lungs of all NNK-treated animals (Figure 3). The showsrate of thegrowth distribution of the tumors of 18F-Nifene increased in a significantlycontrol and NNK- after treatedthe 6th A/J month mice. post-NNK The urinary treatment bladder (Table 18F-Nifene 1). Figure uptake 4 increased over time, while the liver and kidneys retained Figure 3 CT Scans. Representative CT scans of (A) control and (B) NNK-treated AJ mice, showing lung tumors (arrows) 10 months after NNK treatment. (C) Time-course changes of the volumes of lung tumor 18F-Nifene in the lungs of control A/J mice was similar to growth in the NNK-treated AJ mice (data are mean  SD, p 0.05 for thata significant previously amount seen inof BALB/cradioactivity. mice [33],This demonstratingdistribution of 18 indicating lung regions in the same animals were taken based on the that normal lungs have very low F-Nifene uptake. the different age groups). Baseline measures prior to NNK treatment In contrast to control A/J mice, NNK-treated animals 18F-Nifene uptake and contrast and may not necessarily reflect tumors. in vivo studies. Ex vivo studies of the binding in the lungs. Volume of lung tumors correlated control A/J mice lungs showed very little binding of directlyexhibited with a significant time interval amount after ofNNK treatment, showing excised18F-Nifene after (Figure the 5). The lungs from A/J mice treated 18 3 (Figure 3C). Multiple lung tumor nodules F-Nifene retaininga rapid increase 18F after 6 months when the tumor volume binding, which was predominantly localized to the tumor with NNK demonstrated significantly higher PET/CTexceeded images 7 mm (Figure 4). Quantitative analysis of the 18 -Nifene were18F-Nifene confirmed binding by providedthe coregistered tumor/ and amount of F-Nifene binding provided the tumor/ nodules (Figure 5C). Quantitative analysis of the extent nontumor ratios of 1.8-2.0. nontumor ratios 4. extent and amount of 18 18 Ex vivo studies of lung 18F-Nifene uptake Comparative F-Nifene and F-FDG studies 18F-Nifene uptake 18F-Nifene and 18F-FDG for early in the control vs. NNK-treated mice, the lungs were diagnosis of lung tumor in A/J mice. 18F-FDG PET/CT is In order to ascertain the difference in We compared efficacy of Table 1 Quantitation of CT and PET imaging in NNK-treated A/J mice

Study Baseline 3 months 6 months 8 months 10 months Ex Vivo

CT a 0.14 mm3 0.70 mm3 1.57 mm3 na 7.2 mm3 na

18F-Nifene na na na 1.8-2.0b na 4d

18F-FDG na na na 1.5c na 3e

aIn vivo measures of tumor volume in NNK-treated animals (n3, data are mean  SD, p 0.05 for the three age groups); bRatio of tumor/nontumor of 18F-Nifene in NNK-treated animals; cRatio of tumor/nontumor of 18F-FDG in NNK-treated animals; dRatio of tumor/nontumor of ex vivo lung 18F-Nifene at 8 months post treatment; eRatio of Tumor/Nontumor of ex vivo lung 18F-FDG at 8 months post treatment; na: not available. Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137 132

Figure 4 18F-Nifene PET/CT. Representative in vivo 18

F-Nifene PET images of control18F-Nifene (A-C) binding and experimental with CT images. A/J mice 8 months after NNK treatment (D-F). Arrows indicate reciprocal areas in the lungs in control and experiment mice. Transverse CT section (G), PET (H) and coregistered PET/CT image of NNK-treated mouse. Arrows indicate tumors identified by co-localization of an established approach to imaging diagnosis of lung CT scan (Figure 7A-D). The NNK-treated animals clearly cancer [2]. Uptake of 18F-FDG in the fasted control mice occurred in the brain with low amounts in the heart lungs in the in vivo CT (Figure 7E). Uptake of 18F-FDG was seenexhibited in these the presenceregions (Figu of multiple tumor nodules in the adipose tissue uptake of 18F-FDG in NNK-treated mice the PET/CT coregistered image in Figure 7G. The uptake and other peripheral organs (Figure 6). Greater brown re 7F) and was confirmed in 18 kidneywas in keepingand urinary with bladder, the reports 18F-Nifene on the effectswas also of retained nicotine fromin the the heart NNK-treated made it difficult mice, the to exclearly vivo scanvisualize localized some the of inon this the tissue brain, (46). particularly While F-Nifene in the nAChR-rich was excreted thalamus into the the18F-FDG tumor uptake nodules. to lung However, tumors when (Figure the lung7H). wasThe excisedtumor- 18F-FDG in the localized 18F-FDG uptake was not present in the lungs of lung, which was in agreement with the 18F-FDG PET/ control animals. [36]. Control mice showed little uptake of 133 Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137

Figure 5 Ex vivo 18F-Nifene PET/CT. Representative ex vivo 18F-Nifene PET/CT images of A/J control mice (A-C) vs. mice treated with NNK (D-F). Arrows lower amount of 18F-Nifene and the absence of tumors. indicate lung tumors in CT image (D), PET image (E) and the coregistered CT/PET image (F). The images of the control mice (A-C) show significantly

Figure 6 Whole body 18F-Nifene and 18F-FDG imaging studies. Representative in vivo 18F-FDG and 18F-Nifene scans of control and NNK-treated A/J mice. (A) 18 18F-Nifene uptake in control mouse thalamus 18F-FDG uptake in the coronal plane of the control mouse showingF-FDG high uptake activity in controlin the upper mouse chest brain and (BR) heart and and excretion kidneys in(D) the 18F-FDG urinary uptake bladder in the (UB). NNK-treated (B) mouse brain (BR), brown adipose tissue (TH) and excretion in kidneys (K). Nonspecific uptake18 was seen in the harderian glands (HG). (C) 18F-FDG uptake in the coronal plane of the NNK mouse showing high activity in the upper(BAT) andchest excretion and heart. in Note:the urinary 18 bladder (UB). (E) F-Nifene uptake in the thalamus (TH) and excretion by kidneys (K) of the NNK-treated18F-Nifene mouse. is selectivelyNonspecific localized uptake wasin the seen thalamus, in the harderianbrain and lungs.glands (HG). (F) F-FDG uptake is shown in the saggital plane to show the differences in brown adipose tissue binding, while

Overexpression of 4 nAChR in lung tumors of A/J mice Discussion Immunoblots of lung proteins from tumor-free control This study was focused on development and validation of A/J mouse vs. tumor-free and tumor-containing lungs of the PET/CT diagnostic imaging methodology utilizing 18F- Nifene. Previous works have established that 18F-Nifene 4 nAChR subunit in the tumor-containing lung (Figure 42 nAChR [30-32, mice treated with NNK demonstrated overexpression of 18F-Nifene control (not shown) and NNK-treated A/J mice showed canis a visualize highly specific 4-made radioligand nAChRs in ofthe lung tumors of NNK- 8A). Immunofluorescence staining of lung sections of the preferential binding of 4 antibody to cancer cells treated34-36]. Herein, A/J mice, we thus tested enabling the hypothesis early diagnosis that of lung comprising the tumor loci visualized by light microscopy cancer. Imaging of live animals prior to necropsy allowed (Fi 18 nodules retaining F histopathology. The obtained results demonstrated for the 4-madegure 8B,C). nAChRs. These results confirmed that the lung tumor correlation of the tumor load identified by PET/CT and -Nifene expressed large amounts of first time feasibility to visualize lung cancer lesions in live Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137 134

Figure 7 18F-FDG PET/CT. Representative transverse 18F-FDG PET/CT images of control and NNK-treated mice. Designations are as in Figure 4C.

Figure 8 4 nAChR in lung tumors of A/J mice. (A) Immunoblots of lung proteins from tumor-free control mouse (1), and tumor- free (2) and tumor-containing (3) lungs of A/J mice treated with NNK (n  4). The 4 protein band is seen at  Overexpression of  4 in the lung of control mouse. 56 kD. (B) Light microscopy of lung tumor in A/J mouse. (C) Immunofluorescence staining for 4 in a serial lung section at the peripheral tumor area marked out in panel "B". (D) Immunofluorescence staining for A/J mice, and quantitatively characterize lung tumor load and suppresses apoptosis [23, 52-58], indicating that starting from the very beginning of tumor development. nicotine can act as a tumor promoter that facilitates the outgrowth of cells with genetic damage. In addition to a well-formulated etiologic role of tobacco nitrosamines 4 subunit- containingOur findings nAChRs vividly demonstratedbinding 18F-Nifene. that lung This cancer was illustratedcells in A/J by mice immunoblotting abundantly express and corroborated the by in the genotoxic damage inducing lung cancer, recent 4 studies have identified tobacco nitrosamines as high- antibody, and is also in keeping with previous reports of nAChRsaffinity agonists act as of central nAChRs mediators that can upregulate in the activation cell growth of [59]. Therefore, it is currently believed that pulmonary upregulatedimmunohistochemical  studies with specific anti- are considered as novel drug targets for prevention and cancer signaling pathways [60], and these receptors that NNK upregulates4 expression  in nicotine-treated lung cancer cells [26, 47, 48]. Furthermore, it has been reported 4 nAChR [49]. While previous Thetreatment ability of oflung 18 Fcancer-Nifene [61-64]. to detect NNK-induced lung studies of smoker's lungs as well as human and monkey cancer in A/J mice was evident, compared to the very 18 transcriptomesbronchial cells ofexposed tumor-containing to nicotine lungs also demonstrated from the A/J low retentions of F-Nifene in lungs of control mice. overexpression of 7 nAChR [16, 20], the analysis of Postmortem histopathological analysis of the lungs the 7 subunit gene [37]. Therefore, 4 nAChR subunit mice treated with NNK did not confirm overexpression of erence in brain 18F-Nifene binding was observed confirmed the presence of tumors. No significant tumors in the A/J mouse strain. between control and NNK-treated mice. The whole-body appears to be a specific marker of NNK-induced lung distributiondiff of 18F-Nifene in intact mice has been recently It has been demonstrated that activation of nAChRs described by us elsewhere [33]. Since uptake of 18F-Nifene on respiratory cells can contribute directly to lung in the heart is low, delineation of lung tumor uptake was tumorigenesis [50, 51]. Activation of nAChRs stimulates not hampered from spillover of activity from the heart. the growth of lung cancer cells, in part, through 4 nAChR 2,

The tumor/nontumor ratios were approximately 135 Galitovskiy V et al., J Cancer Res Ther 2013, 1(4): 128-137

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Acknowledgement DL, et al. (2005) Receptor-mediated tobacco toxicity: regulation This research was supported by the National Institutes of gene expression through alpha3beta2 nicotinic receptor in oral epithelial cells. Am J Pathol 166:597–613. regulates nicotinic acetylcholine receptors in bronchial epithelial [16] Fu XW, Lindstrom J, Spindel ER (2009) Nicotine activates and up- [17] Wong HP, Yu L, Lam EK, Tai EK, Wu WK, et al. (2007) Nicotine fromof Health American grants LungR01AG029479 Association (JM) to and SG. WeS10RR024546 thank Drs. cells. Am J Respir Cell Mol Biol 41:93–99. (JM) and R01ES017009 (SG) and a research grant promotes cell proliferation via alpha7-nicotinic acetylcholine Harmadeep Dhaliwal, Cristian Constantinescu and M. receptor and catecholamine-synthesizing enzymes-mediated Reza Mirbolooki for their invaluable assistance with

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