mesothelin ( msln ) is a 40-kda cell differentiation - associated glycoprotein appearing with carcinogenesis .
msln was found as an antigen recognized by the monoclonal antibody ( mab ) , k1 , generated by immunization of mice with the human ovarian carcinoma cell line , ovcar-3 .
the protein has been named as msln because the expression of msln in normal tissue was limited to mesothelial cells lining the pleura , pericardium , and peritoneum . on the contrary , msln is widely expressed in human cancers , for example , the majority of ovarian cancers and pancreatic adenocarcinomas , and in 100% of epithelial mesotheliomas .
recent studies showed that it is also found in lung adenocarcinomas , gastric cancers , triple - negative breast cancers , uterine serous carcinoma , acute myeloid leukemia , and cholangiocarcinoma [ 213 ] . because of its limited distribution in normal tissues and elevated expression in cancers , msln has the potential to become a suitable target for a wide range of cancer diagnosis and therapy by using its specific antibodies .
a precursor of msln is encoded as a 622-amino acid glycoprotein and cleaved by furin into a membrane - attached 40-kda form ( msln ) and a 31-kda - shed protein , megakaryocyte potentiating factor ( mpf ) .
the physiological function of msln is not fully elucidated as msln - deficient mice are fertile and do not exhibit any apparent phenotype .
however , recent studies indicate that msln may play an important role in cell adherence , cell survival / proliferation , tumor progression , and chemoresistance .
msln may aid in the peritoneal implantation and metastasis of tumors through its interaction with ca125 ( also known as muc16 ) , an ovarian cancer antigen [ 1618 ] .
msln overexpression promotes cancer cell invasion by inducing matrix metalloproteases 7 and 9 [ 19 , 20 ] .
overexpression of msln constitutively activates nf-b and it leads to higher interleukin-6 production and induces tumorigenesis .
further , the msln expression promotes resistance to certain chemotherapy drugs , such as tnf- , paclitaxel , and a combination of platinum and cyclophosphamide [ 23 , 24 ] .
since msln is overexpressed in a variety of malignancies , it is a good target for anti - msln antibody - based diagnosis and therapy .
a number of anti - msln mabs have been developed , and morab-009 ( amatuximab ) is a chimeric ( mouse / human ) antibody .
hn1   was isolated from a human scfv phage display library and converted into a fully intact human igg . as trace amount of msln
can be detected in the blood of some patients with msln - positive cancers , in vitro diagnostic tests have been developed not only for diagnosis but also for following the course of some of these patients .
a murine mab against msln , clone 11 - 25 , was established by immunizing mice with recombinant human msln .
the 11 - 25 mab was utilized in a sandwich elisa for detecting soluble form of msln in sera of patients with mesothelioma .
the 11 - 25 mab binds to msln in soluble form(s ) and to a membrane - attached form . because the soluble form(s ) of msln is present in very small amount ( 1.43.8  nmol / l )
, it should not interfere with antibody - based therapies that target the msln antigen on cancer cells .
positron emission tomography ( pet ) is a noninvasive , highly sensitive , and a quantitative tomographic imaging modality .
it is clinically important as an imaging tool in cancer diagnosis and staging for a number of malignancies .
the antibody - based pet technology is an attractive method for noninvasive tumor detection since this strategy combines the high sensitivity of pet with the high antigen specificity of mabs .
cu ( t1/2 = 12.7  h ) is the widely used isotope for antibody - based pet , partly due to its wide availability , low cost , and versatile chemistry .
as msln is overexpressed in a wide range of cancers , anti - msln 11 - 25 mab has the potential to become a pet imaging agent for detecting various kinds of msln - expressing cancers . in this study
, we performed in vitro and in vivo investigations of anti - msln ( 11 - 25 ) mab to evaluate its utility as an imaging probe for detecting msln - expressing tumors . to apply to pet imaging
, we labeled dota - conjugated 11 - 25 mab with positron - emitting cu and monitored in vivo distribution through pet imaging of human pancreatic cancer xenografts in nude mice .
mono - n - hydroxysuccinimide ester 1 , 4 , 7 , 10-tetraazacyclododecane-1 , 4 , 7 , 10-tetraacetic acid ( dota - mono - nhs ester ) was purchased from the macrocyclics ( dallas , tx ) .
amicon ultra 0.5 centrifugal filter units were purchased from merck millipore ( billerica , ma ) .
anti - msln mab 11 - 25 ( igg2b ,  ) was prepared from the culture media of hybridoma clone 11 - 25 , which was previously generated by immunizing mice with a recombinant human msln protein , as described .
briefly , balb / c mice were immunized with recombinant msln ( 10571908  bp region of nm_005823 : secretory extracellular domain ) , mixed with freund 's complete adjuvant for first immunization , and boosted 4 times with the same antigen emulsified with the incomplete adjuvant .
the msln - specific antibodies were purified on a protein a column from the culture fluid of hybridoma cells . among the purified mabs , 11 - 25 mab showed the highest reactivity in immunohistochemistry and flow cytometry .
as igg2b isotype - matched control , mouse mab against keyhole limpet hemocyanin ( klh ) was purchased from r&d systems ( minneapolis , mn ) .
human pancreatic cancer cell lines , bxpc-3 , cfpac-1 , and panc-1 , human lung cancer cell lines , nci - h226 , msto-211h , and nci - h520 , and a human epidermoid carcinoma , a-431 , were purchased from american type culture collection ( atcc , rockville , md ) and were maintained in rpmi-1640 ( for bxpc-3 , h226 , 211h , and h520 ) , iscove 's modified dulbecco 's medium ( imdm ; for cfpac-1 ) , and dulbecco 's modified eagle medium ( dmem ; for panc-1 ) , containing 10% fetal bovine serum , 1% penicillin / streptomycin , in a humidified incubator maintained at 37c with 5% co2 .
the concentration of the soluble form of msln , in conditioned culture media of cancer cell lines , was determined by a sandwich elisa in a similar way , as previously reported .
ninety - six - well microtiter plates ( maxisorp , thermo fisher scientific inc . , waltham , ma ) were coated with anti - msln mab , 1430 ( 5  g / ml ) at 4c overnight .
each cell line was cultured for 5 days in their appropriate culture medium and the conditioned medium was collected by centrifugation .
the conditioned medium was diluted five- and tenfold with pbs containing 1% bsa , 0.1% tween-20 , and added to the wells .
after washing with pbs containing 0.1% tween-20 , the wells were incubated for 1 hour with biotinylated mab , 11 - 25 , and subsequently reacted for 30 minutes with avidin - conjugated peroxidase ( dako , glostrup , denmark ) diluted to 1  :  20,000 .
followed by four washes with pbs , 100  l / well tetramethylbenzidine ( tmb ) ( moss inc . ,
soluble msln concentration was calculated by referring to a standard curve using serially diluted recombinant msln .
western blot analysis was performed with cancer cells , that is , bxpc-3 , cfpac-1 , panc-1 , and a-431 . from each cancer cell line ,
total proteins were extracted with ripa buffer ( 25  mm tris - hcl ph 7.6 , 1% np-40 , 0.1% doc , 0.1% sds , 0.15  m nacl , 1  mm edta , 10  mg / l leupeptin , and 1  mm pmsf ) and electrophoresed on 415% sds - polyacrylamide gradient gel ( bio - rad ) .
5% skimmed milk in 20  mm tris - hcl ph 7.5 , 500  mm nacl , 0.05% ( v / v ) tween-20 ( tbs - t ) for 1 hour , the blot membrane was incubated with 10  g / ml of 11 - 25 mab or mouse anti- action mab , ac-15 ( sigma ) , at 4c for 18 hours . the membrane was then incubated with peroxidase - labeled anti - mouse igg f(ab)2 ( rockland , gilbertsville , pa ) for 12 hours at 4c . after washing with tbs - t buffer , the color was developed with dab .
the area of each band was measured with the imagej software ( national institutes of health , bethesda , md ) .
semiquantitative reverse transcription pcr was performed to analyze the expression of msln in bxpc-3 , cfpac-1 , panc-1 , and a-431 cells .
total rna was extracted from cancer cell lines using trizol reagent ( life technologies corporation , carlsbad , ca ) after standard protocol .
msln- and glyceraldehyde-3-phosphate dehydrogenase- ( gapdh- ) mrna were first reverse - transcribed to cdna .
semiquantitative pcr was carried out using the lightcycler real - time detection system ( roche diagnostics , mannheim , germany ) , according to the manufacturer 's instructions , for 50 cycles of 10 seconds ( s ) at 95c , 2  s at 68c , and 10  s at 72c .
cultures of bxpc-3 , cfpac-1 , panc-1 , and a-431 cells were harvested into single - cell suspension by treatment with pbs containing 0.1% collagenase type iv ( invitrogen ) and 1  mm edta .
the 1  10 cells were washed once with cold pbs and incubated for 1 hour on ice in the presence of the 11 - 25 mab at 5  g / ml in pbs containing 2% fbs and 1  mm edta . as control ,
cells were treated with isotype control ( igg2b ) at 4  g / ml . then , the cells were washed twice and incubated with goat anti - mouse igg labeled with alexa fluor 488 for 1 hour on ice .
the cells were washed twice and suspended in 0.5  ml pbs containing 2  g / ml propidium iodide and 1  mm edta before analysis with a facsaria flow cytometer ( bd biosciences ) .
bxpc-3 , cfpac-1 , and panc-1 cells were plated onto 8-well chamber slides at a concentration of 2  10 cells/0.2  ml / well and incubated for 3 days at 37c with 5% co2 .
the cells were washed three times with serum - free medium and fixed with 4% formaldehyde in pbs at room temperature ( rt ) for 10 minutes .
the cells were washed with pbs containing 10  mm glycine and blocked with dako protein block for 10 minutes .
then , the cells were treated with 5  g / ml anti - msln mab overnight at 4c .
the cells were washed with pbs and incubated with fitc - labeled anti - mouse igg ( life technologies ) , alexa fluor 594-labeled wheat germ agglutinin ( wga ) ( life technologies ) , and 4 , 6-diamidino-2-phenylindole dihydrochloride ( dapi ) ( dojindo , kumamoto , japan ) for 30 minutes .
cells were imaged using a confocal laser scanning microscope , lsm510 ( zeiss , oberkochen , germany ) . for staining of cancer xenografts ,
the cancer cells were inoculated to nude mice , as described in the animal model section , and the resulting tumors were taken from the mice , soaked in oct compound , and frozen .
the frozen sections were prepared with cryostat and stained with alexa fluor 488-labeled 11 - 25 mab , alexa fluor 594-labeled wga , and dapi .
all animal experiments were conducted in accordance with guidelines of okayama university and approved by university 's animal care and use committee ( oku-2013098 ) .
five - week - old male balb / c nude mice were purchased from charles river ( tokyo , japan ) and were maintained under specific pathogen - free conditions at okayama university before use . for in vivo
fluorescent imaging , male balb / c nu / nu mice ( 8 - 9 weeks old ) were inoculated subcutaneously with panc-1 ( 1  10 cells ) in the right thigh and bxpc-3 ( 5  10 cells ) in the left thigh . for pet imaging ,
male balb / c nu / nu mice ( 9 - 10 weeks old ) were inoculated with cfpac-1 ( 3  10 cells ) in the right shoulder as well as panc-1 in the right thigh and bxpc-3 in the left thigh .
the imaging experiments were performed when the tumors became about 8  mm in diameter .
the 11 - 25 mab was labeled to alexa fluor 750 dye , according to the manufacturer 's protocols ( molecular probes inc . ,
briefly , the antibody ( 1  mg / ml ) in 0.1  m carbonate buffer ( ph 8.8 ) was reacted with alexa fluor 750 for 1 hour in the dark .
unlabeled dye was removed by gel filtration on a sephadex g-25 ( ge healthcare , uppsala , sweden ) column .
purified alexa fluor 750-labeled antibody was filtrated through a 0.2 m syringe filter and stored at 4c before use .
the absorbance at 280  nm and 752  nm was measured and the degree of labeling ( dol ) was calculated after the manufacturer 's instructions .
alexa fluor 750-labeled 11 - 25 mab ( 90  g / mouse ) was administered via tail vein of mice bearing both bxpc-3 and panc-1 ( n = 3 ) .
fluorescence from the labeled alexa fluor 750 was then monitored by ivis-200 imaging system ( xenogen , alameda , ca ) with emission at 680  nm and excitation at 780  nm 24 , 48 , and 72 hours after intravenous ( i.v . )
dota conjugation to mabs was carried out by incubation in pbs ph 7.0 for 3 hours at a ratio of dota - nhs : 11 - 25 mab , or control anti - klh mab ( igg2b ,  ) , being 100  :  1 .
the number of conjugated dota molecules per molecule of igg was calculated with maldi - tof ms ( 4800 plus maldi tof / tof analyzer , ab sciex , framingham , ma ) by comparison of average molecular mass of untreated and dota - conjugated antibodies .
cu was produced by a cyclotron ( hm-12 cyclotron , sumitomo heavy industries , ltd . , tokyo , japan ) and purified , according to the previously reported method . for radiolabeling ,
cucl2 ( 480550  mbq ) was diluted with 0.1  m sodium acetate buffer ( ph 6.5 ) and added to 0.51  mg of dota - conjugated 11 - 25 mab or 0.48  mg of dota - anti - klh mab , respectively .
radiochemical purity was determined by a combination of thin - layer chromatography and autoradiography ( tlc - arg ) and hplc ( lc-20 , shimadzu co. , kyoto , japan ) . for tlc - arg , samples were spotted on silica gel plates ( silica gel , 60 rp-18 f254s , millipore ) and developed using 50  mm edta ( ph 8.0)/methanol = 1/2 as the mobile phase .
radioactivity derived from antibody - bound cu stays at the origin and unbound cu goes to the top of the chromatogram .
hplc was performed on tskgel super sw3000 column ( 4.6  mm  30  cm , tosoh corp . ,
tokyo , japan ) with 10  mm phosphate 300  mm nacl ( ph 7.0 ) as a mobile phase at a flow rate of 0.35  ml / min . in vitro stability of the radiolabeled mabs after 24 and 48 hours of incubation in mouse plasma at 37c was also analyzed .
cu - dota-11 - 25 mab or anti - klh mab ( 50  l ) was added to 450  l of mouse plasma .
after 24- and 48-hour incubation , aliquots of the incubated mixture of radiolabeled mab with the plasma were injected to hplc with the super sw3000 column , the eluate was fractionated , and their radioactivity was counted by a -counter ( accuflex 7001 , hitachi aloka medical ) .
titers of 11 - 25 mab , dota - conjugated 11 - 25 mab , and cu - dota-11 - 25 mab were tested by elisa .
the wells of a 96-well microtiter plate were coated with 100  l of the purified msln protein at 1  g / ml and incubated overnight at 4c .
wells were blocked with pbs containing 1% bsa for 2 hours . then serially diluted antibody solution was added and incubated for 1 hour .
after three washes with 250  l pbs containing 0.05% tween-20 , the wells were treated with peroxidase - conjugated goat anti - mouse igg ( life technologies ) for 1 hour .
after washes , 100  l of tmb - us ( moss inc . , pasadena , md ) was added and the color was developed .
then , the reaction was stopped by adding 100  l of 2  n h2so4 .
the absorbance at 450  nm was measured with sunrise - basic plate reader ( tecan , grodig , austria ) .
assay was performed with live cells using a modification of the method of li et al . .
bxpc-3 cells were transferred to 24-well plates at 5  10 cells / well / ml and cultured for 4 days in a co2 incubator at 37c .
five to eight hundred nm of cu - dota-11 - 25 mab was incubated with the cell monolayers in triplicate for 2 hours on ice in a complete growth medium ( rpmi-1640 medium containing 10% fetal bovine serum ) .
the cells were washed twice with 1  ml of ice - cold pbs and lysed by incubation at 37c in 0.3  ml of 0.2  m sodium hydroxide for 30  min .
aliquots of each sample were analyzed for protein by a bicinchoninic acid assay ( pierce ) .
dissociation constant in nm was estimated by nonlinear fitting of the specific binding versus the concentration of cu - dota-11 - 25 mab using prism software ( graphpad software , inc . , la jolla , ca ) .
each mouse bearing bxpc-3 , cfpac-1 , and panc tumor was anesthetized by inhalation of isoflurane and injected with approximately 11  mbq of cu - dota-11 - 25 mab ( 15  g mass ) ( n = 3 ) or cu - dota - anti - klh mab ( 11  mbq in 14  g protein ) ( n = 3 ) via the tail vein and 30-minute ( at 0 and 24 hours ) or 60-minute ( at 48 hours ) static pet scans were performed using a small animal pet scanner ( clairvivo pet , shimadzu , kyoto , japan ) and the images were reconstructed using the 3d - drama method . after the pet scans , ct data were acquired at 80  kv and 450  a with slice thickness of 90  m by using explore ct ( ge healthcare japan , tokyo , japan ) .
pet and ct images were converted into dicom format , fused , and analyzed using the pmod software version 3.3 ( pmod technologies ltd . ,
following the terminal pet scans at 48 hours after injection , all mice were euthanized for biodistribution studies .
in addition , separate groups of mice were injected with 11  mbq of cu - dota-11 - 25 mab or with cu - dota - anti - klh mab and euthanized at 24 or 48 hours after injection for biodistribution studies .
the data were expressed as percentage of injected dose per gram of tissues ( % id / g ) . in vivo stability
was measured by tlc . when the mice were sacrificed for biodistribution analysis 24 and 48 hours after injection ,
an aliquot of the blood was taken and centrifuged at 5,000  g for 10  min and 1  l of the plasma was spotted on the tlc .
also an aliquot of liver and kidney was weighed and three volumes ( v / w ) of pbs were added and homogenized .
the homogenate was centrifuged at 5,000  g for 10  min and 1  l of the supernatant was spotted to the tlc plate .
statistical analysis was performed using a nonpaired student t - test for comparison of 2 groups .

 table 1 shows the result of soluble msln determination in the culture media of cancer cells , determined by a sandwich elisa .
pancreatic adenocarcinoma cells , cfpac-1 and bxpc-3 , and lung mesothelioma cells , msto-211h and nci - h226 , were positive for soluble msln . on the other hand ,
pancreatic carcinoma cells , panc-1 , and lung squamous carcinoma cells , nci - h520 , were negative . to determine the expression of the msln protein on the pancreatic carcinoma cells ,
figure 1(a ) shows that bxpc-3 and cfpac-1 cells expressed the msln protein but panc-1 and a-431 cells did not .
a-431 is human epidermal carcinoma cell line and is known as msln - negative .  
figure 1(c ) shows the results of flow cytometric analysis of the four cancer cells with anti - msln mab , 11 - 25 .
the antibody reacted with 50.9% of bxpc-3 cells , 31.4% of cfpac-1 cells , 0.6% of panc-1 cells , and 4.4% of a-431 cells .
next , the expression of msln mrna on the four cell lines was investigated by semi - quantitative pcr ( figures 1(d ) and 1(e ) ) .
the binding of 11 - 25 mab to the fixed cultured pancreatic carcinoma cells and xenografts in mice was examined by fluorescent immunohistochemical staining .
the antibody showed specific binding to both cultured cells and xenografts of cfpac-1 and bxpc-3 cells , but not to those of panc-1 cells ( figures 2(a ) and 2(b ) ) . to analyze the in vivo distribution of 11 - 25 mab by fluorescence imaging , alexa fluor 750-labeled 11 - 25 mab
was administered to mice bearing bxpc-3 and panc-1 tumors , and in vivo imaging was conducted with ivis 200 .  
figure 3 shows the result of nirf imaging . at 24 hours after injection , fluorescence from alexa fluor 750-labeled 11 - 25 mab was detected throughout the body of the animal ; however , msln - positive bxpc-3 xenograft gave off brighter fluorescence than its neighborhood did .
furthermore , time - dependent accumulation of fluorescence to bxpc-3 tumor xenografts was observed and fluorescence in other parts was cleared gradually . at 72 hours after injection , significant fluorescence strength was detected mainly in the bxpc-3 tumor xenograft .  
figure 3(b ) shows the ex vivo image of the dissected tumor xenograft of another mouse treated in the same way at 24 hours after injection .
dota - conjugated 11 - 25 mab or anti - klh mab was purified by column chromatography with pd-10 .
the average molecular weights of 11 - 25 mab and dota-11 - 25 mab determined by maldi - tof - ms were 151,599  da and 153,038  da , respectively .
the average number of dota molecules conjugated per 11 - 25 mab molecule was 3.7 , as determined by dividing the mass difference between dota-11 - 25 mab and 11 - 25 mab by the molecular weight of dota ( 386  da ) . in the same way , the average number of dota molecules per anti - klh mab was determined to be 4.8 .
binding of dota - conjugated 11 - 25 mab to the msln protein showed that 11 - 25 mab had a similar affinity to the msln after conjugation with dota ( figure 4 ) .
dota - conjugated 11 - 25 mab and anti - klh mab were incubated with cu and purified as described in section 2 .
the radiochemical purity of resultant cu - dota - mabs determined by hplc was 97.9% for 11 - 25 mab and 100% for anti - klh mab .
the specific activities of cu - dota-11 - 25 mab and cu - dota - aklh mab were 0.74 and 0.82  mbq/g , respectively . the immunoreactivity retention after radiolabeling of 11 - 25 mab against msln protein was measured by elisa ( figure 4 ) . at the concentration of 1000  ng / ml
, the od value of cu - dota-11 - 25 mab was 66.0% compared to that of native 11 - 25 mab . in vitro stability in plasma after 24 and 48 hours of incubation analyzed by hplc was 78.0% and 76.9% for cu - dota-11 - 25 mab and 82.8% and 77.9% for cu - dota - anti - klh mab .
the same preparations were also analyzed by tlc arg ( figure 5(a ) ) and the results tended to be higher ( 94.3% and 92.6% for cu - dota-11 - 25 mab ) than those analyzed by hplc . for in vitro experiment , cu - dota-11 - 25 mab was prepared and a binding study of cu - dota-11 - 25 mab with alive bxpc-3 cells was performed at 4c .
bxpc-3 cells exhibited saturable cu - dota-11 - 25 mab binding ( figure 6 ) .
the kd of cu - dota-11 - 25 mab binding for the msln on bxpc-3 cells was 353  63  nm . we performed pet imaging of cu - dota-11 - 25 mab in mice bearing bxpc-3 , cfpac-1 , and panc-1 tumors at 0 , 24 , and 48 hours after the intravenous injection .
figures 5(b ) and 5(c ) show the in vivo stability of cu - dota-11 - 25 mab and cu - dota - anti - klh mab in the plasma , liver , and kidney at 24 hours and 48 hours after injection .
both of cu - dota-11 - 25 mab and cu - dota - klh mab were stable in the blood until 48 hours after injection , and they were partly metabolized in liver and kidney extracts . 

figure 7(a ) is the 3d volume rendering image of ct of the mouse and shows the position of xenografts and the position of the cross section . in the upper cross section images of figure 7(b ) , high accumulation of cu - dota-11 - 25 mab was observed in the cfpac-1 xenograft ( red arrowhead ) at 24 and 48 hours . in the lower cross section images , cu - dota-11 - 25 accumulation was observed in the bxpc-3 xenograft ( yellow arrowhead ) but not in the panc-1 xenograft ( white arrowhead ) . on the other hand , the accumulation of cu - dota - anti - klh mab in cfpac-1 and bxpc-3 xenografts ( figure 7(c ) ) was lower than those of cu - dota-11 - 25 mab . in the biodistribution study , relatively high accumulation of cu - dota-11 - 25 mab
was observed in the blood , liver , and msln - positive tumors ( figure 8) . in cfpac-1 xenografts ,
the accumulation of cu - dota-11 - 25 mab was significantly higher than that of cu - dota - anti - klh mab at both time points . in bxpc-3 xenografts ,
the accumulation of cu - dota-11 - 25 mab was significantly higher than that of cu - dota - anti - klh mab at 48 hours after injection ( figures 8(a ) and 8(b ) ) .
at both 24 and 48 hours after injection , the tumor to blood ratio and tumor to muscle ratio of cu - dota-11 - 25 mab in bxpc-3 tumor and in cfpac-1 tumor were significantly higher than those in panc-1 tumor ( p < 0.05 and p < 0.01 , resp . , figures 8(c)8(f ) ) .
the average weight of individual panc-1 , bxpc-3 , or cfpac-1 tumor was 77  49  mg , 50  38/mg , or 207  78  mg , respectively .
in vivo stability of cu - dota-11 - 25 mab and cu - dota - aklh mab was measured by tlc .
the membrane - bound form of msln is present on a wide range of cancer cells [ 213 ] and its expression in normal tissues is relatively limited in the mesothelial cells .
soluble form of msln is found in the circulation in some cancer patients and monitoring the level of soluble msln is useful for diagnosis of malignant pleural mesothelioma [ 26 , 32 ] .
but the levels are too low ( 1.43.8  nmol / l )   to affect the in vivo targeting by anti - msln antibodies . with these properties of msln expression , msln can be a promising targeting molecule for a diverse range of cancers .
our aim is to develop a specific antibody - based pet probe that is available for detecting such a wide range of cancers . for that purpose
, we selected 11 - 25 mab as a candidate and confirmed that 11 - 25 mab was available for detecting msln in western blotting , flow cytometry , immunohistochemistry , and in vivo nirf imaging .
the high mortality rate of the pancreatic cancer is due to the high incidence of metastatic disease at initial diagnosis , the aggressive clinical course , and the lack of adequate systemic therapies .
however , patients diagnosed at an early stage have the potential for therapeutic approach for prolonged survival . there is a need to develop a highly specific technique for early diagnosing .
accumulating evidence has shown that msln is overexpressed in various cancers , including pancreatic adenocarcinoma , ovarian cancer , and mesothelioma .
msln is an attractive candidate as a molecular target for pancreatic cancer marker - specific imaging or immunotherapy .
so , we selected pancreatic adenocarcinoma cells , bxpc-3 and cfpac-1 , as an example set of target cells .
we first confirmed that 11 - 25 mab can detect the soluble form of msln in the conditioned medium of pancreatic adenocarcinoma and mesothelioma ( table 1 ) .
pancreatic adenocarcinoma cells , cfpac-1 and bxpc-3 , excreted soluble msln but epithelioid carcinoma , panc-1 , did not .
then we confirmed the expression of msln protein in these cells by western blot and flow cytometry using 11 - 25 mab and the expression of mrna by semiquantitative reverse transcription pcr ( figure 1 ) .
the results were msln - positive for cfpac-1 and bxpc-3 and negative for panc-1 , which were consistent with the soluble msln levels and previous reports [ 32 , 33 ] .
the immunohistochemistry results of these cell lines detected by 11 - 25 mab showed that msln localized on the membrane and in the cytoplasm ( figure 2 ) . on the culture slides or dishes
, we observed that only a part of bxpc-3 cells , that is , the cells at the edge of cell clusters , was stained with 11 - 25 mab . at the edge of the cluster
it was reported that msln plays a role in cell adhesion , migration , and proliferation [ 3436 ] .
this tendency is similar to the previous immunohistochemistry reports that the expression of msln was strongly observed in the invasive component of pancreatic cancer , but not in the noninvasive component even within the same specimen [ 4 , 37 , 38 ] . on the contrary ,
the xenografts of bxpc-3 and cfpac-1 in mice were uniformly stained with 11 - 25 mab ( figure 2(b ) ) .
part of the reason is supposed to be that the human carcinoma cells were  invading  in mouse tissue and overexpressed msln . as shown in figure 3
, we confirmed the specific accumulation of nirf - conjugated 11 - 25 mab in msln - positive bxpc-3 xenograft by in vivo fluorescent imaging .
as the blood clearance of alexa fluor 750-labeled 11 - 25 mab was slow , the contrast between msln - positive tumors and normal tissues was better at 48 hours after injection than at 24 hours after injection .
nirf has excellent optical characteristics in terms of stability , high sensitivity , and low autofluorescence . although tissue penetration of nirf is limited ( up to several centimeters in tissues )
, nirf - labeled antibody could be used for image - guided surgical resection of tumors in combination with preoperative pet imaging to detect the tumors . for the detection of cancer , high - sensitivity imaging in deep tissue
pet imaging has the potential to meet the required sensitivity for tumors ; besides it is noninvasive and is directed to the whole body .
the radioactive metals are widely used for labeling peptides and antibodies by conjugation of metal chelators .
cu has a half - life of 12.7 hours and decays by 17.9% by + decay to ni , 39.0% by  decay to zn , 43.1% by electron capture to ni , and 0.475% -radiation / internal conversion .
these emissions are 0.579 and 0.653  mev for  and positron , respectively , and 1.36  mev for . cu has been widely used for labeling antibodies for pet [ 4042 ] and several clinical studies revealed that cu - labeled igg can detect tumors within 48 hours after injection [ 43 , 44 ] .
we used mono - n - hydroxysuccinimide ester 1 , 4 , 7 , 10-tetraazacyclododecane-1 , 4 , 7 , 10-tetraacetic acid ( dota - mono - nhs ester ) as a chelating agent for cu .
although dota does not form very stable complex with cu , several clinical studies revealed that cu - labeled dota - igg is adequately stable for pet imaging to detect lesions even around the liver .
many structures , reported to form cu - complexes with improved stability , such as teta , te2a , and cb - te2a , require harsher reaction conditions to incorporate cu .
in addition , although dota - conjugated therapeutic and diagnostic agents , such as y - dota - toc , are clinically approved , any other chelating agents which form cu - complex are not .
the dota-11 - 25 mab had similar reactivity to antigen msln , as compared with native 11 - 25 mab .
further , cu - dota-11 - 25 mab kept the antigen specificity and bound to bxpc-3 cells in vitro with dissociation constant ( kd ) at 353  63  nm ( figure 5 ) . 

cu - dota-11 - 25 mab enabled visualization of msln - positive tumors by pet imaging .
high accumulation of cu - dota-11 - 25 mab in msln - positive tumors was observed compared to that in the msln - negative tumor .
as the tumor size of cfpac-1 tumor was larger than the others , nonspecific accumulation of igg probably by enhanced permeability and retention effect in the cfpac-1 tumor was observed .
the biodistribution studies showed that the accumulation of cu - dota-11 - 25 mab in bxpc-3 and cfpac-1 tumors was significantly higher than that in panc-1 cells ( figure 8) .
however , the clearance of cu - labeled igg in the blood was very slow in general .
reported the biodistribution of in ( t1/2 = 2.8 day)-k1 antibody in athymic nude mice bearing a-431-k5 ( a-431 cells transfected with mesothelin gene ) xenografts .
the peak uptake of in - k1 by a-431-k5 tumors was at 72 hours after injection . as the half - life of cu is shorter than that of in , we only investigated the biodistribution at 24 and 48 hours after injection .
although the tumor to blood ratio was no more than 1 , the ratio of cu - dota - mab in msln - positive tumors was significantly higher than that in msln - negative panc-1 tumor .
the accumulation of cu - dota-11 - 25 mab in the liver was relatively high , as it was previously reported for cu - labeled igg .
the presence of fc receptor might have led to the nonspecific binding of igg into the liver .
transchelation of cu to superoxide dismutase and metallothionein could also be a part of the reason for the accumulation in the liver .
as the distribution of cu - dota-11 - 25 mab in the pancreas or stomach was of very low level ( figure 7 ) , msln - positive pancreatic cancer or gastric cancer could be visualized in high contrast by in vivo pet imaging with cu - dota-11 - 25 mab .
antibodies against human epidermal growth factor receptor 2 ( her2 ) have been approved for the treatment of her2-positive breast cancer and they are used for companion diagnostic testing for targeted cancer therapies , as her2-positive breast cancer is about 30% . on the contrary , msln overexpression has been observed in a wide range of cancers ; diagnostics with pet probe made from anti - msln antibody has the potential to be applicable to a broad spectrum of cancers .
development of fragmented antibody derivatives such as fab and single - chain fv should be useful to achieve faster blood clearance and better  target - to - non - target  contrast at an early time point after administration relative to that of igg 11 - 25 mab . in conclusion ,
11 - 25 mab specifically detected msln both in soluble form by elisa and in cell - attached form by flow cytometry / immunohistochemistry and in vivo nirf imaging .
further , we successfully labeled 11 - 25 mab with cu , with preservation of immunoreactivity .
we evaluated the affinity of cu - dota-11 - 25 mab in vitro and used it as an in vivo pet imaging probe to detect msln - expressed pancreatic cancers .
it was confirmed that cu - labeled 11 - 25 mab significantly accumulated in msln - expressing tumors compared to msln - negative tumor by pet imaging and biodistribution studies .
as msln is highly expressed in various human tumors , our findings suggest that msln - specific imaging using cu - labeled 11 - 25 mab has the potential to be widely used in the diagnosis of patients with msln - positive cancers such as malignant mesotheliomas and ovarian cancers and pancreatic cancers .
 mesothelin ( msln ) is a 40-kda cell differentiation - associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers , including the majority of pancreatic adenocarcinomas , ovarian cancers , and mesotheliomas , while its expression in normal tissue is limited to mesothelial cells lining the pleura , pericardium , and peritoneum . 
 clone 11 - 25 is a murine hybridoma secreting monoclonal antibody ( mab ) against human msln . in this study 
 , we applied the 11 - 25 mab to in vivo imaging to detect msln - expressing tumors . in in vitro and ex vivo immunochemical studies , we demonstrated specificity of 11 - 25 mab to membranous msln expressed on several pancreatic cancer cells . 
 we showed the accumulation of alexa fluor 750-labeled 11 - 25 mab in msln - expressing tumor xenografts in athymic nude mice . 
 then , 11 - 25 mab was labeled with 64cu via a chelating agent dota and was used in both in vitro cell binding assay and in vivo positron emission tomography ( pet ) imaging in the tumor - bearing mice . 
 we confirmed that 64cu - labeled 11 - 25 mab highly accumulated in msln - expressing tumors as compared to msln - negative ones . 
 the 64cu - labeled 11 - 25 mab is potentially useful as a pet probe capable of being used for wide range of tumors , rather than 18f - fdg that occasionally provides nonspecific accumulation into the inflammatory lesions . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion