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CD73-derived adenosine suppresses anti-cancer immunity, and CD73 inhibitors are currently evaluated in several clinical trials. Here, we have assessed enzyme kinetics of all key purinergic ectoenzymes in five cancer cell lines (Hodgkin lymphoma, multiple myeloma, pancreas adenocarcinoma, urinary bladder carcinoma, and glioblastoma) under normoxia and hypoxia. We found that adenosine metabolism varied considerably between individual cancer types. All cell lines investigated exhibited high ecto-adenosine deaminase (ADA) activity, which critically influenced the kinetics of adenosine accumulation. Combining kinetics data with single-cell RNA sequencing data on myeloma and glioblastoma cancerous tissue revealed that purine metabolism is not homogeneously organized, but it differs in a cancer type-specific fashion between malignant cells, stromal cells, and immune cells. Since purine metabolism in cancerous tissue is most likely spatially heterogeneous and differs between the various cell types, diffusion distances in the microenvironment as well as ADA activity may be important variables that influence the level of bioactive adenosine. [Display omitted] • Extracellular adenosine metabolism varies considerably between cancer cell types • Ectoenzyme cascades generating adenosine are not linearly present on cancer cells • Adenosine production from ATP requires enzymes of surrounding non-cancer cells • ADA activity on cancer cells critically influences extracellular adenosine levels Hesse et al. compare the extracellular metabolism of anti-inflammatory adenosine used for immunoevasion in different cancer types. They show that the ectoenzyme cascade generating adenosine from nucleotides is not linearly present on cancer cells but requires enzyme activities of surrounding non-cancer cells for extracellular adenosine production. [ABSTRACT FROM AUTHOR]

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5′-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A 2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A 2A agonist, 4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2 mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3- a ][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3 mg/kg i.p.). Nimesulide (5 mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3 mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5′-(α,β-methylene)diphosphate (APCP; 400 μg/paw). Furthermore, we found increased activity of 5′-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4 h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E 2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A 2A receptors. [ABSTRACT FROM AUTHOR]