The ectonucleotidase enzymes play distinct roles in mammalian organisms by regulating the extracellular concentration of ATP/ADP/AMP/adenosine. The balance of nucleotides/adenosine determines the activation of distinct purinergic receptors triggering specific effects. Like other pleiotropic molecules, the expression of these enzymes may be found in several normal tissues. CD39 and CD73 are constitutively expressed in a variety of tissues and leukocytes and may be upregulated by pro-inflammatory cytokines, hypoxia, and oxidative stress . Regarding normal bladder, the ectonucleotidases play role in nerve-mediated detrusor contractions and in exocytosis of the umbrella cell layer by controlling the ATP/adenosine concentrations in the extracellular space [14–15]. It is possible that CD39 and CD73 are involved in other physiological processes of the lower urinary tract, but this remains uncertain.
In the pathophysiological context, CD39 and CD73 have been found in neoplasias raising the hypothesis of their involvement in tumorigenesis [7, 16]. In the present study, we have shown that the upregulation of CD39 is associated with the non-muscle-invasive form of BC, while upregulation of CD73 with low expression of CD39 is associated with the muscle-invasive form, raising the hypothesis that the nucleotides/adenosine balance may be effective in determining invasive phenotype in BC cells.
With regard to CD39, its expression has not been studied in BC until now. On the other hand, there is some information about CD73 in urothelial tumors. Wettstein and coworkers showed that CD73 predicted favorable prognosis in patients with NMI urothelial BC . In their study, cases of high expression of CD73 were more frequent in pT1 and pTa stages, although no association with CD39 expression was made . More recently, Koivisto and coworkers demonstrated similar results, expanding the analysis to MI BC. They demonstrated that high expression of CD73 by neoplastic cells was more frequent in NMI than in MI BC , a phenomenon not found in our study. In our study, isolated CD73 expression was not sufficient to predict the BC stage (Table 4), suggesting the use of isolated CD39 expression or CD39/CD73 expression balance as a useful strategy to access invasive status of the BC. In other tumors, CD73 has been associated with the worst prognosis. CD73 overexpression promotes invasion, migration, adhesion, and metastasis of human breast cancer cells and in melanomas [19, 20].
Although there is little information about CD39 in BC, it has been widely described in other tumor types. CD39 is overexpressed in pancreatic cancer correlating positively with long-term survival after surgery treatment . In rectal adenocarcinoma, although CD39 was strongly expressed in malignant cells being associated with early tumor stage, its association with CD73 offered a better strategy to predict the prognosis . The combined analysis displaying low CD39 and high CD73 expression in both protein and mRNA levels was associated with invasion and metastasis leading to an unfavorable clinical outcome , similarly as we have proposed herein for BC.
In terms of mechanism, acting sequentially, CD39 and CD73 efficiently hydrolyze extracellular ATP to adenosine, the latter having an effect in down-modulating the antitumor immunity via binding to purinergic receptors. Adenosine-activated A2A receptor protects tumors from antitumor T cells . The blockage of CD73 using a monoclonal antibody enhances antitumor response . Different from adenosine, ATP is a potent pro-inflammatory mediator limiting tumor growth. To promote antitumor immunity, ATP activates inflammasome , and pyroptosis , promotes chemotaxis of monocytes, macrophages, and neutrophils27, and controls tumor-infiltrating CD8 + T cells  and NK cells .
Considering the above, we would propose that the overexpression of CD39 and/or CD73 could increase the adenosine concentration in the extracellular space, which could induce local immunosuppression and consequently tumor progression. However, cases of CD39LowCD73Low and CD39HighCD73High were more frequent in MI and NMI BC, respectively (Table 4). In fact, access to ATP/adenosine status would be critical in understanding this controversial phenomenon. An important limitation of our study is the lack of technical approaches to determine ATP/adenosine in patient tumor specimens. Immunostaining analysis for CD39 and CD73 is a simplistic form to access the complex nucleotides/adenosine system. The final effects will be determined by multiple factors. First, the amount of ATP that moves into the extracellular space, either through cell injury or through channel transport. The tumor microenvironment can present a 1000-fold higher ATP concentration into interstitial space compared to normal tissues . Second, in addition to CD39 and CD73, both nucleotides and nucleosides can be removed from extracellular space by several other ectoenzyme families5. Third, ATP and adenosine exert effects by binding purinergic receptors - P1 receptors, with adenosine as the main ligand, and P2 receptors, with ATP and ADT as the main ligands; P2 receptors are subdivided into seven ionotropic P2X and eight metabotropic P2Y subtypes [31, 32]. Therefore, the roles of ATP/adenosine balance in BC will depend on various expression levels and coexpression patterns.
Finally, our study complements the findings of Stella and coworkers, in which the profile characterized by downregulation of NTPDase 3 and upregulation of CD73 was associated with the late stage of BC , a phenomenon also demonstrated in an animal model . In their hypothesis, that profile establishes an extracellular microenvironment with high levels of ATP and adenosine  favorable to tumor progression .
In conclusion, the altered expression of CD39 and CD73 presented herein supports the idea that these ectonucleotidases are involved in bladder tumorigenesis. Our results suggest that malignant urothelial cells of human BC strongly express CD39 and CD73; however, high expression of CD39 in tumor cells is correlated with the early stage of BC. Further studies are necessary to clarify the effective role of the CD39 and CD73 in the establishment of invasive phenotype in BC cells.