The attenuated growth of tumors in the CD73-deficient mice having increased lymphoid ATPase and ADPase activities is compatible with the possibility that decreased peritumoral ATP concentration is detrimental to the tumor. To study this hypothesis experimentally, we injected melanoma cells into the WT mice, and then treated the tumors locally with apyrase, which hydrolyzes ATP and ADP into AMP. We found that apyrase-treated mice had significantly smaller tumors than vehicle-treated animals Tigecycline (Fig. 3). In addition, the tumor size in apyrase-treated WT mice was not different from those seen in CD73-deficient mice.

Strikingly, apyrase treatment had no effect in tumor-bearing CD73-deficient mice. These data strongly suggest that lowering of the peritumoral ATP

levels either therapeutically by apyrase or genetically by deletion of CD73 effectively inhibits tumor growth. In the apyrase-treated WT mice, the numbers of Tregs (FoxP3+) and MR+and Clever-1+macrophages were lower than in control-treated WT mice (Fig. 5). In fact, the numbers of these cell types in the apyrase-treated WT mice were at a similar level as in the vehicle-treated CD73-deficient mice (also having higher NTPDase activity). Apyrase treatment had no effect on these leukocyte populations in the mice lacking CD73. Moreover, apyrase treatment significantly increased the number of CD8+ T cells in the tumors in both genotypes. Finally, we tested find protocol whether the beneficial effects of CD73 deletion on tumor progression can also be achieved by pharmacological manipulation of CD73 activity. Melanoma-bearing mice were treated peritumorally with a non-hydrolyzable nucleotide analog α,β-methylene-adenosine-5′-diphosphate (AMPCP), which selectively inhibits ecto-5′-nucleotidase. The results showed significant inhibition of tumor growth in WT animals (tumor volume 415±83 in PBS-treated mice and 121±24 mm3 in AMPCP-treated mice respectively, n=3–4 animals/group). AMPCP treatment had no effect

on tumor volume in CD73-deficient mice (tumor volume 150±34 and 150±95 mm3 in PBS- and AMPCP-treated CD73-deficient mice, n=4 mice/group). Thus, chemical inhibition of CD73 activity Interleukin-2 receptor is a therapeutically amenable option to control tumor growth. We have shown here that tumor growth is impaired in CD73-deficient mice. This correlates with diminished intratumoral accumulation of Tregs and macrophages expressing type 2 markers (MR, Clever-1, IFN-γ and NOS2) in the absence of CD73. Lack of CD73 results in increased ATP- and ATP-hydrolyzing activity in immune cells, and we show that by reducing peritumoral ATP levels or by inhibiting CD73 activity in WT mice we can reproduce the CD73-deficient phenotype.