(A) Levels of extracellular adenosine in the TME following 60% oxygen breathing. Microdialysis probes were placed in MCA205 intradermal tumors. After initial probe of the TME, mice were placed in 21% or 60% oxygen for 3h and new probes were placed in the same tumor area. Adenosine levels were measured by reversed-phase liquid chromatography-tandem mass spectrometry using a triple quadrupole mass spectrometer with 13C10-adenosine as an internal standard (P = 0.01) as in (SI, Ref. 7-9). (B) RT-qPCR analysis of CD73 and CD39 in B16 s.c. tumors from mice breathing 21% or 60% O2 for 72 h (P < 0.05, n = 3). (C) Expression of CD73 on the surface of TME-infiltrating T cells. Lymphocytes were isolated from the TME of mice bearing MCA205 pulmonary tumors breathing 21% and 60% oxygen and the expression of CD73 was measured by flow cytometry (CD4, P < 0.02, n = 4; CD8, P < 0.02, n = 4). (D) Breathing 60% oxygen down-regulates immunosuppressive A2AR, A2BR, and COX-2. Mice bearing established MCA205 pulmonary tumors were treated with 21% or 60% oxygen for 72 h and total RNA was extracted and subjected to RT-qPCR analysis (P < 0.05, n = 3).