metastasis to the lung is often a lethal cause in sarcomas and other cancers [ 1 , 2 ] . many attempts to eradicate cancer through conventional therapy ( irradiation or chemotherapy ) are ineffective , mainly because of treatment resistance [ 35 ] . highly metastatic cells tend to exhibit a greater survival ability and resistance to apoptosis than poorly metastatic ones . new imaging procedures for diagnosis , treatment planning , and therapy of metastatic cancer are thus required for accurate staging . several attempts to optimize the methods for imaging lung metastases have been described , such as bioluminescence imaging ( bli ) or fluorescence reflectance imaging ( fri ) of ht-1080 fibrosarcoma cells labeled with luciferase or green fluorescent protein ( gfp ) , respectively [ 6 , 7 ] , and single photon emission computed tomography ( spect ) of prostate cancer cells expressing na+/i - symporter , or fibrosarcoma cells expressing herpes simplex virus type 1 thymidine kinase ( hsv1-tk ) . however , although these models have great potential in preclinical researches , the need of the stable expression of an exogenous marker gene limited their applications in clinic . efficient molecular tracers that can target metastatic tumors through binding an endogenous receptor expressing on cancer cells need to be developed . peptides have several advantages such as small size , good tissue diffusion , target accessibility , no antigenicity , easy synthesis , and adequate radiolabeling . the application of a specific tumor - targeting peptide requires enough binding sites ( e.g. , an overexpressed cell surface receptor ) , high affinity of the ligand , and sufficient in vivo stability . some peptide probes such as somatostatin , gastrin , bombesin , glp-1 , and rgd analog peptides have been extensively examined and applied to diagnosis and radiopeptide therapy . most of these peptides target tumor by recognizing a regulator peptide receptor overexpressed in tumors . for example , somatostatin analogs , the most prominent example of tumor - specific peptide , recognize mainly somatostatin receptor subtype 2 overexpressed in neuroendocrine tumors . bombesin ( also known as gastrin - releasing peptide ( grp ) ) analogs were applied in diagnostic imaging of prostate and breast tumors in clinic [ 13 , 14 ] . rgd motif does not belong to the regulatory peptide family , but is present in extracellular matrix ( ecm ) components like fibrinogen and vitronectin , which interact with integrin receptors on cells . rgd - based peptides were developed to target neoangiogenic endothelial and various types of tumors in preclinical models . the identification of new tracer peptides that can target tumor cells with sufficient specificity and reduced background binding for in vivo imaging remains a challenge . phage display is a powerful technique to identify novel short peptides that can bind to proteins or other macromolecules , with the major advantage of rapidly selecting a target - specific peptide from a vast dna library . phage display technique has also been used to discover numerous novel peptides with specificity to various types of cancer , and some have been further applied in drug delivery and diagnosis researches ( for a recent review , see ) . our lab has previously reported the use of planar -camera for noninvasive imaging of tumor progression and gene therapy in an experimental blood - borne lung metastases model using fibrosarcoma cells expressing hsv1-tk . nevertheless , the imaging relied on the expression of an exogenous reporter gene integrated in tumor cells . in the current research , we intend to develop a peptide tracer that can target fibrosarcoma tumors in vivo without the need of an exogenous marker in tumor cells . biodistribution analysis and noninvasive imaging using planar -camera showed efficient homing and accumulation of the peptide in tumor site in mice . the peptide shows no sequence similarity to other peptides currently known for tumor imaging , but is homologous to a sequence present in the sixth fibronectin type iii domain of a novel protein similar to vertebrate fibronectin type iii domain - containing protein family ( genbank : caq14006.1 ) . the binding of this peptide to cancer cells was inhibited by fibronectin , suggesting that it may have targeted cells through fibronectin - interacting site(s ) . these results recommend the novel peptide as a promising lead structure for tumor - targeting agent . the ng4tl4-tk cell line was constructed from the parental ng4tl4 fibrosarcoma cell line   ( china medical university , taiwan ) by transfecting with packaged virions of a bicistronic retroviral vector containing hsv1-tk gene . ng4tl4-tk cells were cultured in minimum essential medium ( mem ) supplemented with 10% fetal bovine serum ( fbs ) , 100  units / ml penicillin , 10  mg / ml streptomycin , and 2  mm l - glutamine ( all from gibco invitrogen ltd . , female 6-week - old fvb / n mice were obtained from national taiwan university laboratory animal center . ng4tl4-tk cells ( 5  10 ) resuspended in 200  l of pbs were injected subcutaneously into the right limb of the mouse to form tumor . all animal experiment protocols were approved by the institutional animal care and use committee of taipei medical university . the 12-mer peptide library ( ph.d.12 , new england biolabs , ma , usa ) was used for panning process . briefly , 10 cancer cells were collected and fixed with ice - cold glutaraldehyde ( 0.125% in pbs ) for 10 minutes , washed with pbs , blocked with 3% ( w / v ) bsa in pbs , and then incubated with 10 phage clones for 1 hour at room temperature ( rt ) . after extensive washes with pbs , cells were collected by centrifuge , and the bound phages were eluted with 200  l of elution buffer ( 20  mm glycine - hcl , ph 2.2 ) and then neutralized by adding 12  l of neutralization buffer ( 2  m tris - hcl , ph 9.1 ) . the eluted phages were amplified in er2738 bacteria according to the manufacturer 's protocol and used for the next round of panning . the phages selected after the final round were amplified and stored . to identify the dna sequence of the peptide displayed on the phage , single - phage plaques were picked up and amplified for sequencing using a primer corresponding to phage piii gene sequence : 5-ccctcatagttagcgtaacg-3. for in vitro phage - binding assay , 10 cancer cells in suspension were mixed with 5  10 testing phages in pbs for 2  h at 37c . after 5 washes with pbs , bound phages were eluted and neutralized as described . recovered phages were amplified in er2738 bacteria and the titer was determined by standard plaque - forming analysis . for immunofluorescent microscopic examination , cells were seeded on glass coverslips by   10  cells / well in a 24-well plate overnight . the following day , cells were incubated with 5  10  pfu of phages for 1 hour at rt . after 5 washes with pbs , cells were fixed in 2% formaldehyde and blocked with pbs containing 10% fbs . cells were incubated with the primary mouse anti - m13 antibody ( 1  :  3000 ) and then with the secondary fitc - conjugated antimouse antibody ( 1  :  3000 ) , for 1  hr at 37c , respectively . nuclei were counterstained with propidium iodide ( pi ) ( 1  g / ml ) , and the slide was subjected to a fluorescence - activated laser scanning microscope ( tcs sp5 confocal spectral microscope , leica , usa ) . wild - type phage m13 and an untreated sample served as controls .   for in vivo biodistribution analysis , 200  l of pbs containing 10 phages with or without the competitive synthetic tk4 peptide were injected iv into the tumor - bearing mice through tail vein . five minutes after injection , mice were perfused through heart with 50  ml of pbs . tumor and normal organs including kidney , liver , lung , and heart were removed . after homogenization , 100  g of each tissue was washed with pbst for 3 times and incubated in 200  l of elution buffer at rt with gently shaking for 10 minutes . recovered phages were neutralized and titered by plaque - forming assay and normalized as pfu per gram of tissue . after centrifuge , the supernatant was collected and measured for protein concentration using protein assay kit ( bio - rad , usa ) and then diluted in the coating buffer ( 0.1  m nahco3 , ph 8.6 ) . microtiter wells ( coster , usa ) were coated with 0.5  g / well of cell lysate or bsa in coating buffer at 4c overnight and then blocked with 3% ( w / v ) bsa in pbst at 37c for 1  hr . 10 of testing phage was added to the wells and incubated at rt for 2 hours . after 5 washes with pbst , 50  l of diluted ( 1  :  1000 ) hrp - conjugated anti - m13 antibody ( ge healthcare bio - sciences , nj , usa ) was added to each well and incubated at rt for 1 hour . after 5 washes with pbst , each well was incubated with 50  l of tmb substrate solution ( kpl , md , usa ) in the dark for 30 minutes of development at rt . the reaction was stopped by adding 1  n hcl ( 25  l / well ) , and the plate was read using a microplate reader at 450  nm . cancer cells ( 10 ) were collected and fixed with 2% formaldehyde and blocked with 1% ( w / v ) bsa in pbs . testing phages ( 5  10  pfu ) were incubated with cells for 2 hour at 37c , followed by 5 washes with pbs . the bound phages were labeled by incubating with a primary mouse anti - m13 antibody ( 1  :  3000 ) ( ge healthcare bio - sciences , nj , usa ) for 1  hr at 37c and then with a secondary fluorescein isothiocyanate ( fitc ) conjugated antimouse antibody ( 1  :  3000 ) ( sigma - aldrich , usa ) for 1  h at 37c . the fluorescent signal of each cell was analyzed by facscan flow cytometry ( becton dickinson , nj , usa ) . the synthetic 12-mer amino acid peptide tk4 ( satthyrlqaan ) was purchased from kelowna international scientific inc . ( taipei , taiwan ) , with fitc conjugated on the n - terminal ( fitc - tk4 ) . for in vitro binding assay , cells were seeded on coverslips in 24-well plate by 10  cells / well . the following day , cells were washed by pbs and incubated in serum - free medium containing fibronectin ( sigma - aldrich , usa ) in a concentration 0 , 2 , or 20  g / ml for 30  min at 37c . mediums were then removed , cells were washed by pbs and then incubated in serum - free medium containing fitc - tk4 ( 40  m ) for 1  hr at 37c . medium was removed , and cells were washed 3 times in pbs for 5  min . cells were fixed in 4% pfa for 10  min at rt and then mounted on slides by mounting medium containing dapi ( vector lab , ca , usa ) and examined on a fluorescent microscope ( bx41 , olympus , japan ) . for biodistribution analysis and in vivo imaging , synthetic fitc - tk4 was further labeled on the benzene group of fitc with isotopic i ( [ i]-fitc - tk4 ) . the [ i]-fitc - tk4 used in animal experiment was solved in pbs at a concentration of 50  g/100  ci/100  l . mice were pretreated with 0.5  ml of 0.9% nai ( w / v ) solution by intraperitoneal injection 15  min before [ i]-fitc - tk4 administration to diminish thyroid uptake of the liberated i coming from [ i]-fitc - tk4 . each mouse was then iv injected with 50  g of [ i]-fitc - tk4 ( 100 ci ) dissolved in 100  l of pbs . briefly , static images were obtained from anesthetized animals at 1 , 4 , and 24  hr with a digital -camera ( elscint sp-6 , haifa , israel ) , equipped with a high - energy pinhole collimator , a 364-kev 6 10%i photopeak energy window , and a 256  256  16  bit image matrix . the mice injected with i ( 100  ci ) solution served as negative control . for biodistribution analysis , tumors and organs were removed and weighed , and the radioactivity was determined using a -counter . results were calculated as the percentage of injected dose per gram ( %  id / g ) of tissue . tumors removed from sacrificed mice were also dissected and imaged using ivis-200 optical system ( xenogen , ca , usa ) for the detection of fitc signal . for the selection of a peptide with specific binding affinity to fibrosarcoma cell line ng4tl4-tk , a phage library expressing variant surface 12-mer peptides was used for in vitro biopanning . for each round , 10 phages were incubated with 10 cancer cells for 1  hr and the unbound phages were washed off . the bound phages were then eluted from cancer cells and amplified in bacteria for the next round of panning . after 4 panning rounds , single - phage clones were selected and amplified , and the phage dna was sequenced . among 15 clones sequenced , 11 ones ( 73% ) showed the same peptide sequence : satthyrlqaan ( named tk4 in the following text ) ( table 1 ) . the phage expressing tk4 as well as the synthetic tk4 peptide were used for further evaluation . the tk4-displaying phage ( tk4-phage ) was analyzed for the binding affinity to ng4tl4-tk fibrosarcoma cells . after extensive washes , the bound phages were eluted from cells and titered by a standard plaque - forming assay . the results showed that only the phage clone displaying tk4 peptide was substantially eluted from ng4tl4-tk cells ( figure 1(a ) ) . the binding of tk4-phage to ng4tl4-tk cells was further confirmed by flow cytometry analysis using an anti - m13 antibody ( figure 1(b ) ) . immunofluorescent ( if ) microscopic examination with the same anti - m13 antibody detected the attachment of tk4-phage , but not the wild - type phage , on the surface of ng4tl4-tk cells ( figure 1(c ) ) . to confirm that the binding affinity of tk4-phages to cell did not result from the random absorption on cell membrane , tumor cells were lysed and coated on microtiter wells by 0.5  g of protein per well . after extensive washes , the phages retaining in the well were detected using an hrp - conjugated anti - m13 antibody and quantified by measuring od 450  nm after substrate incubation . the result showed that tk4-phages adhered strongly to tumor lysate but not to control peptide , while the wild - type m13 phages adhered to neither ( figure 1(d ) ) . in summary , these results showed that tk4-phage bound to ng4tl4-tk fibrosarcoma cells and suggested that the binding was associated with one ( or more ) specific cell - surface protein receptor(s ) .   to investigate the tumor targeting of tk4-phage in vivo , phages were injected into tumor - bearing mice intravenously ( iv ) through tail vein . phages were recovered from the homogenates of removed tumor or other organs and were titered by a standard plaque - forming assay . the result showed a significant increase of tk4-phage titer in implanted tumor up to 10-fold in comparison with other organs ( figure 2(a ) ) . in contrast , cp1 control phage did not show any preferential targeting . to confirm that the tumor homing of tk4-phage is dependent on the displayed tk4 peptide , the tk4-phage titers eluted from tumors reduced dramatically after incubation with 100  g of tk4 peptide ( figure 2(b ) ) . these results showed that tk4-phage performed the in vivo tumor - homing ability , which was dependent on the displayed tk4 peptide . to examine the availability of tk4 peptide as an in vivo tumor - imaging probe , the synthetic tk4 peptide was labeled with fitc and isotopic i ( [ i]-fitc - tk4 ) , and iv injected to mice through tail vein . to diminish the thyroid accumulation of liberated i , mice were pretreated with 0.9% nai solution intraperitoneally . whole - body images were taken on a planar -camera 1 , 4 , and 24  hrs postinjection . the results showed that at 4  hr , tk4 signal was detected in tumor site , which then diminished and became unrecognizable in 24  hrs ( figure 3(a ) ) . tumors were also removed from mice sacrificed at 4 or 24  hrs , dissected , and imaged for fitc signal using fri ( figure 3(b ) ) . the results confirmed a much stronger accumulation of tk4 peptide inside the tumor as compared to surrounding muscle . biodistribution analysis of injected peptide was performed by measuring the radioactivity in tumor and other organs removed from sacrificed mice ( figure 4 ) . the peptide uptake in tumor was 3.28  0.63% , 2.66  0.66% , and 1.03  0.76%  id / g at 1 , 4 , and 24  hr postinjection , respectively . although most organs showed higher or comparable radioactivities comparing to tumor at 1  hr , most of them reduced rapidly at 4  hr , while tumor retained most signal ( > 80% ) ( figure 4 ) . the uptake of tk4 was higher in tumor than in other organs at 4  hr except kidney , liver , and intestine ( table 2 ) . kidney and intestine exhibited considerable retention of radioactivity : 36.82  4.31% and 12.02  5.45% at 4  hr , respectively , suggesting that the [ i]-fitc - tk4 was eliminated through both hepatobiliary and renal eliminations . the %  id / g ratio of tumor - to - muscle was 5.73 and 3.15 at 4 and 24  hr , respectively . mice injected with i ( 100  ci ) served as negative control , which showed no specific accumulation in -camera images or biodistribution analysis ( figure 3 , data not shown ) . to understand whether tk4 shows sequence similarity to other peptides currently known for tumor targeting , we have searched in different protein databases , and the results showed no such similarity . instead , the alignment using protein blast in ncbi found a high similarity between tk4 and a novel protein similar to vertebrate fibronectin type iii domain - containing protein family ( genbank : caq14006.1 ) found in danio rerio ( zebrafish ) . the tk4 homologous sequence locates at the sixth fibronectin type iii domain ( fn3 ) of the protein ( figure 5(a ) ) . fibronectin is one of the major fractions of ecm that mediates interactions between cells , other ecm components ( e.g. , collagen ) and other fibronectin molecules . to test if tk4 could bind to fibronectin - interacting site(s ) , we performed an in vitro binding assay of tk4 on ng4tl4-tk cells . the results showed that tk4 binding was inhibited by pretreatment of cells with fibronectin in a dose - dependent manner ( figure 5(b ) ) . these results implied a relationship between tk4 and fibronectin and would suggest that the binding of tk4 to cancer cells is associated with fibronectin - interacting components . drug - resistant metastatic tumors as well as side effects are major limitations of conventional cancer therapy such as irradiation or chemotherapy [ 4 , 5 ] , which necessitate the search for novel tumor - targeting agents . specific targeting of tumors , compared with earlier less specific approaches , is increasingly significant in oncology either for diagnostic or therapeutic purposes . our previous report has demonstrated the monitoring of tumor progression and gene therapy using noninvasive planar -camera imaging in a lung metastases model with murine fibrosarcoma cells , ng4tl4-tk , stably expressing hsv1-tk . following the previous work , in the current research , we are attempting to develop a new targeting agent which can label the same tumor cells in vivo without the need of the expression of an exogenous marker gene in tumor cells . using the peptide phage display system on ng4tl4-tk cell line , tk4-phage was confirmed for the binding affinity to ng4tl4-tk cells in vitro with different methods including plaque - forming assay , flow cytometry , if microscopy , and phage elisa ( figure 1 ) . iv administration of tk4-phage to mice bearing ng4tl4-tk tumors through tail vein showed significantly higher retention of phages in tumor than in other organs ( figure 2 ) . as competitive synthetic tk4 peptide was premixed with tk4-phage , the phage titer eluted from tumors reduced to a level comparable to the control tissue . these results confirmed that tk4-phage maintained its tumor - targeting ability in vivo and that the binding is dependent on the displayed surface tk4 peptide . these results recommended the potential of tk4 peptide as an in vivo tumor - targeting agent . radio- and fluorescent - labeled tk4 ( [ i]-fitc - tk4 ) was thus synthesized and systemically administrated in tumor - bearing mice . although a strong and unspecific distribution of tk4 signal was observed throughout the body 1  hr after injection , most signals diminished rapidly at 4  hr , while the tumor retained a comparable intensity , resulting in a high tumor - to - organ ratio ( figure 4 and table 2 ) . the uptake of tk4 was higher in tumor than in normal organs except kidney , liver , and intestine at 4  hr , and the tumor - to - muscle ratio of %  id / g was 5.73 ( table 2 ) . the weak accumulation of radioactivity in collateral tissues made the diagnostic imaging of tumor more viable . the tumor site can be detected in a whole - body imaging using planar -camera at 4  hr postinjection ( figure 3(a ) ) , and the fri confirmed a much stronger fitc signal accumulated in tumors than in surrounding muscles ( figure 3(b ) ) . all together , these results suggest that tk4 peptide is able to target and accumulate in fibrosarcoma tumors in vivo . the difference in biodistributions between the phage particles and synthetic tk4 peptide was noticed ( figures 2 and 4 ) . the phage particles showed an efficient binding only in tumor , while synthetic peptide had a high retention also in liver and kidney . phage particles were recovered from tissues of sacrificed animal in a few minutes after administration , which may prevent the further accumulation of phages in excretion route like liver , intestine , or kidney . in addition , the whole body perfusion in sacrificed mice may have helped to wash off the unbound phages . to optimize the use of tk4 as an efficient tumor - imaging peptide ; however , the substantial accumulation of radioactivity in kidney and liver should be reduced . small changes in peptide sequence , linkers , chelator , and isotope can dramatically affect the biodistribution . for example , a high radioactivity retention in liver and intestine could result from the hepatobiliary elimination . strategies including glycosylation or conjugation with hydrophilic amino acid have been developed to reduce the liver uptake of rgd peptides ( for a recent review , see ) . renal excretion is a preferable elimination pathway of radiopharmaceuticals and/or their metabolites from the body . nevertheless , high kidney retention may obscure tumor imaging and result in potential radiation nephrotoxicity , and thus limit the use of radiolabelled peptides in medical applications [ 21 , 22 ] . current methods for kidney protection include coadministration of basic amino acids , the bovine gelatin - containing solution gelofusine or albumin fragments , which interfere with the tubular reabsorption pathway of peptides ( for a recent review , see ) . these and other researches have led to developments of more ideal peptide probes for tumor imaging and therapy , and the knowledge can be expanded to further modification of tk4 . our current research showed that tk4 is homologous to a fn3 domain in a novel protein similar to vertebrate fibronectin family and that the tk4 binding on cancer cells can be inhibited by fibronectin ( figure 5 ) , suggesting that tk4 may have targeted tumors via fibronectin - binding site(s ) . it contains an array of repeating domains ( fn1 , 2 , and 3 ) that mediate interactions between cells , other ecm components ( e.g. , collagen ) , and other fibronectin molecules . the tumor - targeting motif rgd , which serves as a common binding site for a variety of integrins , has been originally derived from the tenth fn3 repeat in fibronectin . besides rgd , other peptide sequences have been exploited from fn domains with cell - binding activity such as the essential 4 1 adhesion motif ldv , the 25-mer endothelial cell adhesion peptide cs1 , and the mesenchymal stem cell - adhesive peptide alngr that interacts with integrin 1 . these studies would suggest that due to the pivotal role of fibronectin in the complex ecm architecture , novel binding motif residing in fn sequences or their homologous domains , like tk4 , may be still discovered . we have recently found that tk4 binds efficiently to human lung cancer cell lines and that the binding can be also inhibited by fibronectin pretreatment ( data not shown ) . thus one of our future works will be to evaluate the potential of tk4 as a multitumor - targeting probe . identifying the cell surface receptor(s ) of tk4 on tumor cells , certainly , is crucial and should be performed in the future . in conclusion , our data presented here showed that tk4 , a novel peptide isolated from phage display library , showed a homology to fn3 domain and can target fibrosarcoma tumor cells in vivo after systemic administration . tk4 holds promise as a lead structure useful for selective delivery of therapeutics or diagnostic probe to fibrosarcoma or other tumors .
a 12-mer amino acid peptide satthyrlqaan , denominated tk4 , was isolated from a phage - display library with fibrosarcoma tumor - binding activity . in vivo biodistribution analysis of 
 tk4-displaying phage showed a significant increased phage titer in implanted tumor up to 10-fold in comparison with normal tissues after systemic administration in mouse . 
 competition assay confirmed that the binding of tk4-phage to tumor cells depends on the tk4 peptide . 
 intravenous injection of   131i - labeled synthetic tk4 peptide in mice showed a tumor retention of 3.3% and 2.7%  id / g at 1- and 4-hour postinjection , respectively . 
 tumor - to - muscle ratio was 1.1 , 5.7 , and 3.2 at 1- , 4- , and 24-hour , respectively , and tumors were imaged on a digital -camera at 4-hour postinjection . 
 the present data suggest that tk4 holds promise as a lead structure for tumor targeting , and it could be further applied in the development of diagnostic or therapeutic agent .