Extracellular nucleotides and nucleosides mediate diverse signaling effects in virtually all organs and tissues. Most models of purinergic signaling depend on functional interactions between distinct processes, including (i) the release of endogenous ATP and other nucleotides, (ii) triggering of signaling events via a series of nucleotide-selective ligand-gated P2X and metabotropic P2Y receptors as well as adenosine receptors and (iii) ectoenzymatic interconversion of purinergic agonists. The duration and magnitude of purinergic signaling is governed by a network of ectoenzymes, including the enzymes of the nucleoside triphosphate diphosphohydrolase (NTPDase) family, the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, ecto-5'-nucleotidase/CD73, tissue-nonspecific alkaline phosphatase (TNAP), prostatic acid phosphatase (PAP) and other alkaline and acid phosphatases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). Along with "classical" inactivating ectoenzymes, recent data provide evidence for the co-existence of a counteracting ATP-regenerating pathway comprising the enzymes of the adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK/NME/NM23) families and ATP synthase. This review describes recent advances in this field, with special emphasis on purine-converting ectoenzymes as a complex and integrated network regulating purinergic signaling in such (patho)physiological states as immunomodulation, inflammation, tumorigenesis, arterial calcification and other diseases. The second part of this review provides a comprehensive overview and basic principles of major approaches employed for studying purinergic activities, including spectrophotometric Pi-liberating assays, high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analyses of purine substrates and metabolites, capillary electrophoresis, bioluminescent, fluorometric and electrochemical enzyme-coupled assays, histochemical staining, and further emphasizes their advantages, drawbacks and suitability for assaying a particular catalytic reaction.

Adenosine is an evolutionary ancient metabolic regulator linking energy state to physiologic processes, including immunomodulation and cell proliferation. Tumors create an adenosine-rich immunosuppressive microenvironment through the increased release of ATP from dying and stressed cells and its ectoenzymatic conversion into adenosine. Therefore, the adenosine pathway becomes an important therapeutic target to improve the effectiveness of immune therapies. Prior research has focused largely on the two major ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase 1/cluster of differentiation (CD)39 and ecto-5′-nucleotidase/CD73, which catalyze the breakdown of extracellular ATP into adenosine, and on the subsequent activation of different subtypes of adenosine receptors with mixed findings of antitumor and protumor effects. New findings, needed for more effective therapeutic approaches, require consideration of redundant pathways controlling intratumoral adenosine levels, including the alternative NAD-inactivating pathway through the CD38-ectonucleotide pyrophosphatase phosphodiesterase (ENPP)1-CD73 axis, the counteracting ATP-regenerating ectoenzymatic pathway, and cellular adenosine uptake and its phosphorylation by adenosine kinase. This review provides a holistic view of extracellular and intracellular adenosine metabolism as an integrated complex network and summarizes recent data on the underlying mechanisms through which adenosine and its precursors ATP and ADP control cancer immunosurveillance, tumor angiogenesis, lymphangiogenesis, cancer-associated thrombosis, blood flow, and tumor perfusion. Special attention is given to differences and commonalities in the purinome of different cancers, heterogeneity of the tumor microenvironment, subcellular compartmentalization of the adenosine system, and novel roles of purine-converting enzymes as targets for cancer therapy. Significance Statement The discovery of the role of adenosine as immune checkpoint regulator in cancer has led to the development of novel therapeutic strategies targeting extracellular adenosine metabolism and signaling in multiple clinical trials and preclinical models. Here we identify major gaps in knowledge that need to be filled to improve the therapeutic gain from agents targeting key components of the adenosine metabolic network and, on this basis, provide a holistic view of the cancer purinome as a complex and integrated network.