extracellular adenosine concentration increases under metabolically stressful conditions , notably in the tumor microenvironment , where hypoxia is frequently given [ 2 , 3 ] . such accumulation of adenosine mediates , through four distinct receptors ( a1 , a2a , a2b , and a3 ) , complex and diverse effects that lead to tumor immunoescape .
this includes cytoprotection and growth promotion of tumor cells [ 5 , 6 ] , angiogenesis increase [ 7 , 8 ] , and suppression of effector ( antitumor ) t cells .
although cells are provided with adenosine transporters , the main source of this nucleoside in the tumor interstitium is the hydrolysis of extracellular atp , which also accumulates in tumors , by membrane enzymes known as ecto - nucleotidases [ 6 , 10 ] .
different families of these enzymes , acting extracellularly , are responsible for the generation of adenosine from adenine nucleotides ( i.e. , atp , adp , or amp ) : ( 1 ) the ectonucleoside triphosphate diphosphohydrolase ( e - ntpdase ) family , that includes four plasma membrane - bound members : ntpdase1 ( cd39 ) , ntpdase2 , ntpdase3 , and ntpdase8 ; these enzymes are differentially expressed and hydrolyze with different affinities nucleoside triphosphates and diphosphates to their monophosphate derivatives ( e.g. , atp and adp to amp ) ; ( 2 ) the ectonucleotide pyrophosphatase / phosphodiesterase ( e - npp ) family , capable of hydrolyzing nucleoside triphosphates to monophosphates and pyrophosphate ( ppi ) , such as atp to amp and ppi ; ( 3 ) the alkaline phosphatase ( ap ) family , that includes ubiquitous enzymes degrading broad range of substrates , such as adenine nucleotides and ppi , releasing inorganic phosphate ( pi ) ; ( 4 ) the 5-nucleotidase family , with only one member attached to the outer plasma membrane , the ecto-5-nucleotidase ( cd73 ) , a glycosyl phosphatidylinositol - linked membrane - bound glycoprotein that efficiently hydrolyses amp to adenosine [ 1113 ] .
two members of ecto - nucleotidases families , the e - ntpdase cd39 and the 5-nucleotidase cd73 , acting sequentially , seem to have a crucial role in tumor - immune cell interaction .
they both are expressed not only by infiltrating immune cells but also by tumor cells , and their expression is regulated by hypoxia [ 10 , 14 ] . increased cd39 and cd73 expression has been described in various cancer types , mostly in correlation with a poor prognosis [ 1517 ] .
both molecules are considered promising therapeutic targets in oncology , and cd73 has already been proven to inhibit tumor growth and metastasis in a breast cancer model in mice [ 1820 ] . however , until now there were not available data concerning ecto - nucleotidases expression in endometrial cancer ( ec ) .
there are two clinicopathological variants : the estrogen - related , type i , endometrioid carcinoma , and the nonestrogen - related , type ii , nonendometrioid carcinoma .
although there are different molecular alterations that have been already identified in ec , with different prevalence between tumors [ 22 , 23 ] , there is need to decipher the complete molecular profile of ec pathogenesis to improve diagnosis and favor the design of new therapeutic strategies .
the aim of the present work was to study the expression of cd39 and cd73 in endometrioid ( type i ) and serous ( type ii ) ec when compared with nontumoral endometrium . to achieve this objective , protein and gene expression experiments , as well as in situ enzyme activity assays ,
the ethical principles of this study adhere to the declaration of helsinki , and all the procedures were approved by the ethics committee for clinical investigation of bellvitge hospital .
endometrial samples from adenocarcinoma ( endometrioid and serous types ) and their corresponding nontumoral tissue ( if present ) were obtained from hysterectomy specimens at the service of gynecology of bellvitge hospital .
fresh samples were cut , embedded in o.c.t . freezing media ( tissue - tek ; sakura finetek , zoeterwoude , the netherlands ) , snap - frozen in a shandon histobath 2 ( neslab instruments inc . , usa ) at the service of pathology , and stored at 80c until used .
alternatively , endometrial samples were obtained from the tumor bank of bellvitge biomedical research institute ( idibell ) .
fifteen endometrioid adenocarcinomas ( 13 grade 1 , 2 grade 2 ; all figo stage i ) ( 5682 years old , median 61 ) and fourteen serous adenocarcinomas ( grade 3 ; 9 figo stage i , 2 figo stage ii , and 3 figo stage iv ) ( 6386 years old , median 77 ) , and their adjacent nontumoral endometrium were used in this study .
touch preparations of endometrial cancer tissue samples were obtained by lightly pressing the freshly cut tumor surface on clean glass microscope slides , thus generating a tumor cell imprint .
briefly , tissue sections of 10  m thick and touch preparations were fixed in 10% phosphate - buffered formalin mixed with cold acetone ( merck , darmstadt , germany ) for 2.5 minutes .
fixed samples were rinsed with pbs and preincubated for 1 hour at room temperature ( rt ) with pbs containing 20% normal goat serum ( gibco , paisley , uk ) and 0.2% gelatin ( merck ) .
samples were then incubated overnight at 4c with the following primary antibodies : anti - human cd39 clone bu61 ( ancell corporation , minnesota , mn , usa ) at 1/500 , mouse monoclonal anti - human ecto-5-nucleotidase ( cd73 ) clone 4g4 ( abcam , cambridge , uk ) at 1/50 , and rabbit monoclonal anti - human cytokeratin 19 ( ck19 ) clone epr1579y ( abcam ) at 1/200 .
after three washes in pbs , samples were incubated for 1 hour at rt with the appropriate secondary antibodies : horseradish peroxidase - conjugated goat anti - mouse ( envision + system ; dako , carpinteria , usa ) , alexa fluor 488- or 555-goat anti - mouse or anti - rabbit ( life technologies , paisley , uk ) .
nuclei were counterstained with haematoxylin or , alternatively , in fluorescence assays , to - pro-3 or dapi ( life technologies ) were used to visualize the nuclei .
samples were mounted with fluoromount aqueous mounting medium ( sigma - aldrich , sant louis , missouri , mo , usa ) . the results were observed and photographed under a light leica dmd 108 microscope ( leica microsystems , wetzlar , germany ) or , in fluorescence assays , under a nikon eclipse e-800 microscope ( nikon , tokyo , japan ) or under a leica tcs - sl spectral confocal microscope ( leica ) .
results from both tissue samples and touch preparations were independently evaluated by at least two observers .
label intensity was scored as negative (  ) , intermediate ( + ) , or strongly positive ( + + ) . for enzyme histochemistry ,
adpase and ecto-5-nucleotidase ( ampase ) activities were localized by using the wachstein / meisel lead phosphate method [ 24 , 25 ] in tissue samples and in touch preparations .
briefly , fixed samples were preincubated for 1 hour at rt in 50  mm tris - maleate buffer , ph 7.4 containing 2  mm cacl2 and 0.25  m sucrose .
enzyme reaction was carried out for 1 hour at 37c in the same buffer supplemented with 5  mm mncl2 , 2  mm pb(no3)2 , 3% dextran t250 , and 2.5  mm levamisole , as inhibitor of alkaline phosphatases , and in the presence of 200  m adp or 1  mm amp , as substrate . for cd39 and cd73 inhibition experiments ,
1  mm nf279 ( tocris bioscience , bristol , united kingdom ) and 1  mm  , -meadp ( sigma - aldrich ) were added , respectively , to both preincubation and enzyme reaction buffers .
the reaction was revealed by incubating with 1% ( nh4)2s v / v for exactly 1 minute .
samples were counterstained with haematoxylin , mounted with fluoromount aqueous mounting medium ( sigma - aldrich ) , and observed and photographed as described above .
50100  g of human tumor ( endometrioid and serous endometrial adenocarcinoma ) and nontumoral tissue samples were homogenized in a buffer containing 20  mm hepes , 250  mm sucrose , 0.3  mm pmsf , 1  mm dtt , 1  mm egta , and 1  mm mgcl2 ( ph 7.4 ) using a glass homogenizer ( vidrafoc , barcelona , spain ) .
after homogenization , samples were centrifuged at 600  g for 10 minutes at 4c in a beckman ja-20 centrifuge .
the pellet was discarded and supernatants were centrifuged at 48,000  g for 20 minutes at 4c in a beckman ti-70 centrifuge .
the resulting pellets were resuspended in a buffer containing 20  mm hepes , 0.3  mm pmsf , and 1  mm dtt ( ph 7.4 ) .
protein concentration was determined by the method of lowry et al .   using bovine serum albumin as a standard .
adpase and ampase activities were determined by measuring the amount of pi using the malachite green colorimetric assay , as previously described .
total rna from endometrial tumor tissue samples was isolated using the rneasy plus mini kit ( qiagen , hilden , germany ) , following the manufacturer 's protocol .
total isolated rna ( 2  g ) was reversely transcribed into complementary dna ( cdna ) using the first strand cdna synthesis kit ( fermentas , thermo scientific , chicago , il , usa ) .
quantitative real - time pcr ( qrt - pcr ) was performed to examine the expression of cd39 , ntpdase2 , and cd73 genes .
designed large - scale taqman low - density array ( tlda ) microfluidic cards ( applied biosystems , foster city , ca , usa ) were used .
the 384 wells of each card were preloaded with predesigned fluorogenic taqman probes and primers for cd39 , ntpdase2 , and cd73 .
cdna ( 1  g ) combined with taqman 2x universal pcr master mix ( applied biosystems ) were loaded into each sample - loading port .
qrt - pcr reactions were carried out using the abi prism 7900ht real - time pcr system ( applied biosystems ) .
data were collected using the sds v2.1 software ( applied biosystems ) and analyzed by the comparative ct ( ct ) quantification method using the expression suite v1.0 software ( applied biosystems ) .
the relative expression levels of cd39 , ntpdase2 , and cd73 genes were determined using 18s mrna as an endogenous control for normalization .
results are expressed as the mean of the relative quantification ( rq ) of the tested transcripts ( n = 7 serous adenocarcinoma samples ; n = 7 endometrioid adenocarcinoma samples )  the standard error of the mean ( sem ) .
results were obtained from five independent experiments performed using 1  g of cdna , all with duplicate measurements .
student 's t - test was used to compare the means of two independent groups of normally distributed data .
extracellular adenosine in tumors , mainly generated by the sequential action of ecto - nucleotidases , has immunosuppressive effects through a broad range of actions , including inhibition of antitumor t - cell function , modification of local interleukin levels , and inhibition of phagocytosis ( reviewed in ) . in this section
we show and discuss our results on the expression of the ecto - nucleotidases cd39 and cd73 in type i and type ii endometrial carcinomas .
cd39 was immunolocalized in the stroma of both nontumoral and tumoral endometria ( figure 1(a ) ) .
for the nonpathological endometrium the expression of cd39 has already been previously described in association with stromal cells and blood vessels .
here we show that label score was significantly higher for both endometrioid and serous types of tumors when compared with the corresponding nontumoral coexisting endometrium ( figure 1(b ) ) .
no cd39 labeling was found in endometrial adenocarcinoma epithelia , as demonstrated with the double staining performed with the anti - cd39 and anti - ck19 antibodies ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/509027 ) .
strong in situ adpase activity was detected in the tumor stroma , coinciding with the cd39 immunolocalization ( figure 2(a ) ) .
equivalent results were obtained using atp as substrate for the in situ activity assay ( not shown ) .
adpase activity measured in tumor tissue homogenates demonstrated that serous ( grade 3 ) adenocarcinomas had significantly higher activity than endometrioid ( grade 1 ) adenocarcinomas ( figure 2(b ) ) .
cd73 expression was strongly immunodetected in both types of tumors , in epithelial structures and in the stroma ( figure 3(a ) ) , thus partially colocalizing with ck19 ( supplementary figure 2 ) .
specific cd73 activity , demonstrated with the inhibitor  , -meadp , matched the immunolabeled structures ( figure 3(a ) ) .
we have already previously demonstrated that the expression and activity of cd73 are abundant in nonpathological endometrium [ 24 , 25 ] .
consequently , due to the high expression of cd73 in tumoral and nontumoral endometria , label score comparisons were not possible .
moreover , no differences among tumors were observed with the enzyme assays either in tissue slices or in tissue homogenates ( not shown ) .
immunolabeling and in situ enzyme activity results obtained with the touch prep technique were equivalent to those obtained with tissue slices ( figures 2(c ) and 3(b ) ) .
the usefulness of the touch prep technique for diagnosis has already been demonstrated in breast cancer with 100% sensitivity and specificity in the evaluation of tumor margins at the time of the surgery .
this technique has also been previously used to demonstrate by immunolabeling a decreased expression of p2x7 atp receptor in endometrial cancer cells   and also , recently , the relationship between p53 expression and the tumor grade . however , to our knowledge , this is the first report validating the use of touch preps for enzyme activity studies , therefore opening the possibility of performing such studies in cytological samples . in order to determine
if the differences in cd39 protein and adpase activity between tumor types also involved gene expression changes , quantitative real - time pcr analyses were performed ( figure 4 ) .
cd39 gene expression was 2-fold higher in serous endometrial adenocarcinoma than in endometrioid , coinciding with the data obtained with the protein ( figure 4(a ) ) .
these gene expression changes did not apply to ntpdase2 ( figure 4(b ) ) , indicating that cd39 upregulation is not a general feature of other members of ntpdase family .
no changes were detected in cd73 gene expression between the two tumor types ( figure 4(c ) ) .
these results on endometrial tumors add to the list of human cancers in which cd39 is overexpressed and support the growing body of evidence that cd39 is a potential therapeutic target for cancer immunotherapy .
antibody - based therapy and pharmacological approaches against cd73 have been reported to significantly inhibit tumor growth and improve antitumor immunity in mouse models [ 28 , 32 ] .
this also coincides with the higher grade of these tumors , but further studies are needed to establish statistical correlations with the tumor grade in the case of type i endometrioid tumors .
the consequences of this high cd39 activity in endometrial tumors are increased levels of amp , the substrate for cd73 , and also highly expressed in these tumors , which will , in turn , generate increased immunosuppressive levels of extracellular adenosine .
 one of the strategies used by tumors to evade immunosurveillance is the accumulation of extracellular adenosine , which has immunosupressive and tumor promoting effects . 
 the study of the mechanisms leading to adenosine formation at the tumor interstitium are therefore of great interest in oncology . 
 the dominant pathway generating extracellular adenosine in tumors is the dephosphorylation of atp by ecto - nucleotidases . 
 two of these enzymes acting sequentially , cd39 and cd73 , efficiently hydrolyze extracellular atp to adenosine . 
 they have been found to play a crucial role in a variety of tumors , but there were no data concerning endometrial cancer , the most frequent of the invasive tumors of the female genital tract . 
 the aim of the present work is to study the expression of cd39 and cd73 in human endometrial cancer . 
 we have analyzed protein and gene expression , as well as enzyme activity , in type i endometrioid adenocarcinomas and type ii serous adenocarcinomas and their nonpathological endometrial counterparts . 
 high levels of both enzymes were found in tumor samples , with significantly increased expression of cd39 in type ii serous tumors , which also coincided with the higher tumor grade . 
 our results reinforce the involvement of the adenosinergic system in cancer , emphasizing the relevance of ecto - nucleotidases as emerging therapeutic targets in oncology . 