angiogenesis , the formation of new blood vessels , plays an important role in tumor growth , local invasiveness , and metastatic progression .
many preclinical studies and clinical trials confirm the importance of integrin v3 in the process of tumor angiogenesis and metastasis [ 1 , 2 ] .
integrin v3 is a cell adhesion molecule and is highly expressed on activated endothelial cells and tumor cells but is not expressed on resting endothelial cells and therefore specific for neoangiogenesis .
several extracellular matrix ( ecm ) proteins like vitronectin , fibrinogen , and fibronectin interact with integrin v3 via the amino acid sequence arg - gly - asp ( rgd ) [ 4 , 5 ] .
vascular endothelial growth factor ( vegf - a ) also plays an important role in both normal vascular tissue development and tumor neovascularization and is highly expressed in various human tumors [ 6 , 7 ] .   in the last few years metal pet isotopes such as ga and
both ga and cu are excellent alternatives to f. ga can be obtained from an in - house commercially available ge / ga generator .
the advantage of using cyclotron produced cu for pet imaging and radiotherapy is the longer half - life allowing imaging at late time points acquiring additional information .
cu can now be produced in high yield and at high specific activity on a small biomedical cyclotron .  
radiolabeled rgd peptides for positron emission tomography ( pet ) imaging , targeting integrin v3 in tumors , have been described in several studies with mainly f ( t1/2 = 110  min ; emax ,  + = 634 ( 97% ) kev ) , ga ( t1/2 = 68  min ; emax ,  + = 1.90 ( 89% ) kev ) , and cu ( t1/2 = 12.7  h ; emax ,  + = 653 ( 18% ) kev ) [ 917 ] . for ga and cu different chelating systems have been used .
some of the latest described tracers have used the chelator 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid ( nodaga ) combined with c(rgd ) labeled with ga [ 9 , 12 , 15 , 18 ] or cu [ 12 , 18 ] .
net is one of the tumor types where antiangiogenesis treatment of patients seems promising   but patients need to be selected individually . to select the patients , pet scans with a tracer targeting tumor - angiogenesis
may be extremely relevant . therefore using a human net xenograft model for preclinical studies to evaluate new pet tracers targeting tumor - angiogenesis
the aim of the present study was therefore , as the first , to use and evaluate the pet tracer cu - nodaga - c(rgdyk ) using a human net xenograft model .
biodistribution , pet / ct scans , correlation between gene expression of angiogenesis markers and tracer uptake , and radiation dosimetry extrapolation for humans were performed .
since investigation of tumor - angiogenesis using human xenografts in mice is challenged by the fact that the formation of new vessels in the tumors might be of either human or mice ( host ) origin , we designed qpcr assays to quantify both mice and human genes of integrin v , integrin 3 , and vegf - a .
nodaga - c(rgdyk ) was purchased from abx gmbh ( advanced biochemical compounds , radeberg , germany ) .
cucl2 was produced at risoe technical university of denmark , roskilde , denmark . for high - performance liquid chromatography ( hplc )
analysis a dionex p580 pump with a pda-100 detector and an in - line scansys radioactivity detector was used .
nodaga - c(rgdyk ) ( 2  nmol ) dissolved in 450  l ammonium acetate ( 0.1  m , ph 8.47 ) was mixed with 50  l of cucl2 ( 5060  mbq ) for 15  min at room temperature .
hplc analysis was performed with a jupiter column , 4u proteo 90a , 250  4.6  mm ( phenomenex , torrance , ca , usa ) and flow 1.5  ml / min .
the hplc mobile phase was solvent a , 0.1% trifluoroacetic acid in water , and solvent b , 0.1% trifluoroacetic acid in acetonitrile .
gradient : 02  min 15% b , 27  min 1540% b , and 710  min 4015% b. the retention time of cu - nodaga - c(rgdyk ) was 5.9  min .  
the stability of cu - nodaga - c(rgdyk ) was investigated in either buffer or plasma from nude mice . for buffer stability , 160  mbq cu - nodaga - c(rgdyk ) was incubated for 1 , 2 , 18 , and 24 hours at room temperature in 450  l ammonium acetate buffer . for the stability in plasma , 10  mbq cu - nodaga - c(rgdyk ) was incubated for 1 , 2 , 18 , and 24 hours in 500  l plasma .
the stability of cu - nodaga - c(rgdyk ) in plasma and buffer was analyzed and determined by the previously described hplc conditions .
human lung bronchus carcinoid , nci - h727 obtained from atcc ( american type culture collection , manassas , va , usa ) , was used .
before taken into experiments , the cell line was tested free of mycoplasma at statens serum institute , copenhagen , denmark .
cells were cultured in rpmi ( roswell park memorial institute ) medium 1640 with glutamax ( gibco , life technologies , ny , usa ) containing 10% fetal calf serum ( biological industries ( bi ) , kibbutz beit - haemek , israel ) , and 1% penicillin - streptomycin ( gibco , life technologies ) in 5% co2 at 37c .
female nmri ( naval medical research institute ) nude mice ( 6 weeks upon arrival ) were acquired from taconic europe , lille skensved , denmark .
the mice were acclimated one week in the animal facilities before taken into experiments . at the age of 8 weeks ( weight : 32.8  4.2  g ) the mice had h727 cells ( 57  10 cells in 100  l medium mixed with 100  l matrigel basement membrane matrix ( bd sciences , san jos , ca , usa ) inoculated subcutaneously on the left and right flank during anesthesia with 1  :  1 v / v hypnorm ( janssen pharmaceutica nv , beerse , belgium)/dormicum   ( roche , basel , switzerland ) .
tumors were then grown for 3 - 4 weeks ( average tumor volume 210  mm ) .
animal care and experimental procedures were performed under the approval of the danish animal welfare council ( 2006/561 - 1124 ) .
tumor bearing mice were anesthetized with 3% sevoflurane ( abbott scandinavia ab , solna , sweden ) mixed with 35% o2 in n2 .
mice were tail - vein injected with 2.0  0.7  mbq cu - nodaga - c(rgdyk ) .
biodistribution of cu - nodaga - c(rgdyk ) was obtained at 1 , 2 , and 18 hours after injection ( n = 5 mice in each group ) .
mice were sacrificed by neck drawing and tumors , organs ( liver , kidneys , lung , spleen , heart , intestine , and muscle ) and blood were collected , weighted , and radioactivity measured in a gamma counter ( 2480 wizard , perkin elmer , ma , usa ) .
tx , usa ) , stored overnight at 4c , rnalater then removed , and tumors stored at 80c until total rna extraction and further qpcr experiments were performed .
for estimation of the human radiation dose using cu - nodaga - c(rgdyk ) the mouse biodistribution data were used . assuming similar pharmacokinetics in mice and humans , the activity in organs at 1 , 2 , and 18 hours post injection ( p.i . )
was used to calculate single exponential effective half - lives and residence times for each organ .
these data were then used for extrapolation to human dosimetry estimates using standard female ( 58  kg ) and male ( 70  kg ) human phantoms and the organ level internal dose assessment / exponential modeling software ( olinda / exm ; vanderbilt university , nashville , tn , usa ) . to ensure a conservative estimate ,
the effective dose for both adult male and female were then calculated using olinda / exm .
a longitudinal dynamic study was performed scanning a mouse at 1 , 2 , and 18 hours after intravenous injection of 1.6  mbq cu - nodaga - c(rgdyk ) , allowing dynamic information of the tracer biodistribution in tumors as well as in the other organs of interest .
the pet scans were performed using a small animal pet scanner ( micropet focus 120 , siemens medical solutions , knoxville , tn , usa ) . the energy window for the emission pet scans was set to 350650  kev and the time resolution was 6  ns .
the acquired data for emission scan was stored in list - mode format and postprocessed to obtain 2 bytes 128  144  32 sinograms .
finally , the emission sinograms were reconstructed using map algorithms and resulted into 4-byte 256  256  95 image sets with a zoom factor of 1.443 and a voxel size of 0.87  0.87  0.79  mm .
the small animal ct scans of the mouse parallel with the pet scans were acquired using a small animal computed tomography ( microcat ii , siemens medical solutions ) .
the acquisition time of each ct scan was 6.5 minutes generating 360 projections at 360 arc .
the x - ray source settings were 75  kvp , 500  a , and 270  ms for the voltage , the current , and the exposure time , respectively .
the ct projections on the 3,000  2,970 ccd crystal were binned by 4 to increase sensitivity and reduce the dataset size .
the projections were reconstructed by real - time reconstruction algorithm ( the cobra toolbox ) using shepp - logan filter into 768  768  512 matrix with a voxel size of 0.092  0.092  0.092  mm .  
the fusion of the pet and ct images was performed manually using the asipro toolbox ( siemens medical solutions ) . also
the measurement of the activity uptake in the tumors and the organs were estimated by manually outlining on ct images .
the outlining of the organs on ct images in every slice resulted in 3d region of interest ( roi ) .
the results of these rois acquired the concentration of the tracer in the organs expressed in percentage of the injected dose per gram tissue ( % id / g ) .
the whole tumors were lysed and homogenized in precellys-24 ( bertin technologies , montigny , france ) in tubes containing ceramic beads .
total rna was isolated using nucleospin rna l kit ( macherey - nagel gmbh & co. kg , dren , germany ) .
rna concentration was determined by nanodrop 1000 ( thermo fisher scientific , de , usa ) .
rna quality expressed as rna integrity number ( rin ) was measured on a 2100 bioanalyzer ( agilent technologies , ca , usa ) .
0.3  g total rna was reverse transcribed using affinityscript qpcr cdna synthesis kit ( stratagene , ca , usa ) .  
human itgav , human itgb3 and human vegfa were tested in a triplex- , mice itgav and mice itgb3 in a duplex , and mice vegfa in a simplex - qpcr assay .
the three human housekeeping genes hprt , ubc , and rplp were also measured in a triplex .
the brilliant qpcr core reagent kit ( stratagene ; cat # 600530 ) was used for all the multiplex assays .
assay optimization resulted in a 50% and 100% increase of taq polymerase and dntp in duplex and triplex assays , respectively .
no - rt ( no reverse transcription ) for all the samples was tested using the housekeeping gene - triplex .
the thermal profile for qpcr core reagent was : denaturation at 95c for 10 minutes , followed by 40 cycles with denaturation at 95c for 30 seconds and annealing / elongation at 60c for 1 minute .
the thermal profile for brilliant iii reagent was : denaturation at 95c for 3 minutes , followed by 40 cycles with denaturation at 95c for 20 seconds and annealing / elongation at 60c for 20 seconds .  
the samples were run in triplicates using 1  l of cdna in a total volume of 25 and 20  l , respectively .
the optimal housekeeping genes for the h727 tumors were found by testing 12 human reference genes from tataa biocenter ( goteborg , se ) in 12 different h727 tumors from nmri nude mice . with genorm software   the genes and the number of genes giving the most stable endogenous normalization
the six genes of interest m - itgav ( nm_008402 ) , m - itb3 ( nm_016780 ) , m - vegfa ( nm_001025250 ) , h - itgav ( nm_002210 ) , h - itgb3 ( nm_000212 ) , and h - vegfa ( nm_001025366 ) , along with the housekeeping genes h - hprt ( nm_000194 ) , h - ubc ( nm_021009 ) and h - rplp ( nm_001002 ) were designed using beacon designer ( premier biosoft , ca , usa ) . before designing ,
all genes were tested for cross homology against the human or the mouse genome and checked for secondary structures . for the optimized conditions of all primers and probes
all primers and probes were purchased from sigma - aldrich ( st . louis , mo , usa ) .
qbaseplus software   based on the comparative method ( 2 )   for relative quantification was used including normalization to three housekeeping genes .
furthermore an inter - run calibrator for correction between pcr runs within the same gene and an arbitrary overall calibrator for comparison of the gene levels in between mice and human genes were taking into account .
as shown in earlier studies   the pcr data were found not to be normal distributed when tested by a one - sample kolmogorov - smirnov test .
all qpcr data were therefore log10 transformed to obtain normal distribution and the log - transformed data were used for all the subsequent statistical analyses .  
t - test ( two tailed ) was used for comparison of mrna levels of each gene  human versus mice  at the three different sample collecting times .
all statistical analyses were performed using spss 17.0 statistical software ( spss inc . , chicago , illinois , usa ) .
nodaga - c(rgdyk ) was labelled with cu in 15 minutes at room temperature with a radiochemical purity of 92.596.6% and a specific activity of 25.125.7  mbq / nmol .
the purity of cu - nodaga - c(rgdyk ) in buffer was more than 93% at 1 , 2 , 18 , and 24 hours after incubation .
the purity was also more than 93% after 24 hours when 4.5  ml of saline was added to 500  l of cu - nodaga - c(rgdyk ) solution . in plasma , the purity of cu - nodaga - c(rgdyk ) was 88% after 24 hours of incubation .
rin ( rna integrity numbers ) were measured to be between 6.8 and 9.9 , verifying fine rna quality with limited degradation .
no - rt for each sample was checked in qpcr , finding no ct values , indicating that genomic dna is not present and therefore not disturbing the gene expression values . in h727 tumors ( removed 2 hours after tracer injection )
integrin v of both human and mice origin was expressed at the same level , whereas gene expression of h - vegf - a was significantly higher than m - vegf - a ( p < 0.001 ) .
integrin 3 of both human and mice origin was expressed on a very low level ( figure 2 ) .   regression analyses for cu - nodaga - c(rgdyk ) tracer uptake , % id / g ( calculated from the biodistribution data ) versus quantitative mrna in the h727 tumors showed significant correlation at 2  h p.i . with mice integrin v ( r = 0.76 , p < 0.05 ) , mice integrin 3 ( r = 0.75 , p < 0.05 ) , and mice vegf - a ( r = 0.81 , p < 0.05 ) , also for human integrin v at 2  h p.i .
liver , kidneys , lung , spleen , heart , intestine , muscle , and blood were collected from 10 mice at the following time points : 1 , 2 , and 18 hours post injection .
% id / g was calculated from the measured counts by taking counting efficiency of the -counter ( f = 0.0943 ) , half - life time for cu , decay from injection to counting time , and tissue weight into account .
we found the highest % id / g ( mean  sem ) at 1  h p.i .
for the kidneys , intestines , liver and spleen ( 2.2  0.11 , 1.60  0.16 , 1.05  0.17 , and 0.94  0.11 ) decreasing already at 2  h p.i . (
0.97  0.03 , 0.64  0.04 , 0.65  0.04 , and 0.62  0.04 ) and decreasing further at 18  h p.i .
( 0.51  0.02 , 0.43  0.04 , 0.43  0.02 , and 0.33  0.02 ) .  
tumor - to - blood ratio was 12.7 , 37.2 , and 20.2 , respectively at the 3 collecting times .
residence times based on biodistribution data are shown in table 3 and were used for calculating estimates of human radiation - absorbed doses using olinda / exm .  
table 4 shows the olinda / exm estimates for human adult females and males for each organ and the total effective dose .
the highest radiation - absorbed doses were found to be the urinary bladder wall ( 0.014 and 0.010  mgy / mbq for females and males , resp . ) .
however , this estimate is a worst - case scenario since we used a conservative model with a urinary elimination fraction of 75% with voiding intervals of 5 hours .
the whole body effective dose was 0.038 and 0.029  msv / mbq for females and males , respectively .
accordingly , an administered dose of 200  mbq cu - nodaga - c(rgdyk ) to humans would lead to a radiation burden of less than 8  msv .
the tumors were clearly visible after injection of cu - nodaga - c(rgdyk ) at all measured time points : 1 , 2 and 18  h pi ( figure 5 ) .
cu - nodaga - c(rgdyk ) showed relatively high uptake in h727 tumors ; mean % id / g of right and left tumors were 1.15 , 0.99 , and 0.76 respectively , at 1 , 2 , and 18  h pi .
tumor - to - muscle ( t / m ) ratios were 6.9  0.8 .
both % id / g and t / m ratios were similar to the values found in the biodistribution study using the -counter .
tracer activity in organs did not disturb the tumor to background contrast from 2  h pi where most of the nonspecific uptake had been cleared .
to the best of our knowledge this is the first study of the pet tracer cu - nodaga - c(rgdyk ) .
the major finding was a close correlation between tumor uptake of cu - nodaga - c(rgdyk ) and the target integrin v3 validating its ability to image neo - angiogenesis .
the cyclic rgd peptides c(rgdfk ) and c(rdgyk ) have frequently been used for radiolabeling .
c(rgdfk ) , cyclo [ arg - gly - asp - d - phe - lys ] only differs from c(rgdyk ) , cyclo [ arg - gly - asp - d - tyr - lys ] , in one amino acid , d - phenylalanine ( d - phe ) instead of d - tyrosine ( d - tyr ) .
d - tyr is more hydrophilic than d - phe and c(rgdyk ) has therefore been reported as preferable due to faster renal excretion . in our study
we found a faster decrease of radioactivity in kidneys , intestine , and liver from 1 to 18  h ( 2.2 to 0.5 , 1.6 to 0.4 , and 1.1 to 0.4%id / g ) than that reported for cu - nodaga - c(rgdfk ) in the same time intervals ( 2.2 to 1.6 , 2.1 to 1.3 , and 1.7 to 1.0%id / g ) .
c(rgdyk ) , the peptide used in the present investigation , has previously been chelated to 1,4,7,10-tetraazacyclododecane - n , n,n,n-tetraacetic acid ( dota ) or 1,4,7-triazacyclononane-1,4,7-triacetic acid ( nota ) and labeled with cu [ 1113 , 25 ] or ga .
cu - dota - c(rgdyk ) was first described in 2004 by chen and coworkers .
the cu - dota complex seemed more unstable than cu - nota or cu - nodaga , because cu was transferred to copper - binding proteins , leading to relatively high blood and liver uptake .
nodaga is , compared to the chelators dota and nota [ 8 , 11 , 2729 ] , particularly useful due to high hydrophilicity leading to rapid renal excretion avoiding radioactivity accumulation in the gastrointestinal tract   and in the kidneys .
nodaga - c(rgdy / fk ) has been labeled with ga [ 9 , 12 , 15 , 18 ] and nodaga - c(rgdfk ) also with cu .
one paper compared ga- and cu - nodaga - c(rgdfk ) and found the highest tracer uptake using ga - nodaga - c(rgdfk ) compared to cu - nodaga - c(rgdfk ) ( 5.2 versus 3.8%id / g , 1  h p.i . in
highly angiogenic u87 mg xenograft tumors ) but also 3 times higher tumor - to - blood ratio .
we found similar t / b ratio ( 12.7 ) after 1 hour using cu - nodaga - c(rgdyk ) as they did with cu - nodaga - c(rgdfk ) .
the advantage of cu labeling is the possibility of delayed imaging up to 12 or 18  h post injection with expected improved image contrast , and therefore improved visualization of low expressing integrin v3 tumors .
indeed , we found that delayed images after 2 and 18 hours showed higher tumor - to - background ratios compared to 1 hour images .
in addition cu has a much better image quality than ga due to the shorter positron range .
a comparison to f - labeled rgd shows that tumor - to - liver and tumor - to - kidney ratios are similar . for the neuroendocrine h727 xenograft tumors we found moderate levels of tracer uptake ( 1.2%id / g 1  h p.i . as the highest ) explained by the fact that neo angiogenic activity in h727 tumors , reflecting low aggressiveness in general of net tumors , is known to be lower than , for example , in glioblastoma u87 mg xenograft tumors showing high expression of integrins .  
we investigated the target specificity of the pet tracer by analyzing mrna levels of both integrin v and integrin 3 using quantitative qpcr .
our results strongly support that cu - nodaga - c(rgdyk ) has high affinity to integrin v3 reflecting neo angiogenesis .
a challenge in preclinical studies using human xenografts in mice is , that the formation of new vessels in the tumors might be of both human and mouse origin .
the interaction between mouse host stroma and human tumor cells and the role they play in the tumor - angiogenesis and tumor growth are of great importance in antiangiogenesis treatment .
therefore in preclinical studies using antiangiogenic drugs for treatment , it is of great importance to take into consideration species specificity .
accordingly , the antibody bevacizumab only binds to human vegf - a and not to murine vegf - a , whereas the peptide cilengitide is blocking integrin v3 of both human and mouse origin . in the present study we therefore quantified both the gene expression level of mice and human integrin v , integrin 3 , and vegf - a in our human h727 xenograft model .
we found that integrin v and integrin 3 , of both mouse and human origin , are present at comparable levels .
this seems to be true throughout tumor development as we previously ( unpublished data ) found dual origin from 13 weeks of tumor growth with a confirmed higher level of vegf - a of human origin .
species specificity of our pcr designs were verified targeting human or mice gene sequences during blast tests , and as a further control qpcr primers targeting mice genes were tested in cdna from a net patient finding no gene expression .  
the finding of correlation between tracer uptake of cu - nodaga - c(rgdyk ) and mrna levels of mouse integrin v , integrin 3 , and vegf - a two hours post injection indicates that cu - nodaga - c(rgdyk ) is indeed targeting the tumor - angiogenesis and therefore is a promising radiotracer for pet imaging and quantification of angiogenesis .  
taken together cu - nodaga - c(rgdyk ) seems promising for future use in humans .
we therefore performed experiments to estimate dosimetry in humans based on projections from animal data .
here we found a favorable estimate of less than 8  msv for a realistic clinical dose of 200  mbq .
compared to recent dosimetry estimates for ga - nodaga - c(rgdyk )   on a per mbq basis the radiation burden is comparable especially taking into consideration the more conservative urinary elimination model used in our calculations . in both studies
we , as the first , have described the use of cu - nodaga - c(rgdyk ) as a pet tracer targeting angiogenesis .
we found an excellent tumor - to - background ratio due to rapid elimination from nontumor tissue . also an excellent image quality was obtained using cu .
in addition , our compound seems stable as indicated by the low liver uptake . using gene expression analyses we found a close correlation with key angiogenesis markers validating the target of the tracer .
we therefore suggest that cu - nodaga - c(rgdyk ) should be further studied as a promising candidate for human angiogenesis pet .
 
 purpose . 
 the purpose of this paper is to evaluate a new pet tracer 64cu - nodaga - c(rgdyk ) for imaging of tumor angiogenesis using gene expression of angiogenesis markers as reference and to estimate radiation dosimetry for humans . procedures . 
 nude mice with human neuroendocrine tumor xenografts ( h727 ) were administered 64cu - nodaga - c(rgdyk ) i.v . for study of biodistribution as well as for dynamic pet . 
 gene expression of angiogenesis markers integrin v , integrin 3 , and vegf - a were analyzed using qpcr and correlated to the tracer uptake in the tumors ( % id / g ) . from biodistribution data human radiation - absorbed doses were estimated using olinda / exm . 
 results . 
 tumor uptake was 1.2%id / g with strong correlations between gene expression and tracer uptake , for integrin v  r = 0.76 , integrin 3  r = 0.75 and vegf - a r = 0.81 ( all p < 0.05 ) . 
 the whole body effective dose for humans was estimated to be 0.038 and 0.029  msv / mbq for females and males , respectively , with highest absorbed dose in bladder wall . 
 conclusion . 
 64cu - nodaga - c(rgdyk ) is a promising new angiogenesis pet tracer with potential for human use . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion 
5. Conclusion