Assessment of ATP Metabolism to Adenosine by Ecto-Nucleotidases Carried by Tumor-Derived Small Extracellular Vesicles

Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression.

Here, the ATP pathway of ADO production (ATP◊ADP◊AMP◊ADO) by ecto-nucleotidases carried in sEV was evaluated by a novel method using N⁶-etheno-ATP (eATP) and N⁶-etheno-AMP (eAMP) as substrates. The "downstream" N⁶-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL).

Human melanoma cell-derived TEX (MTEX) metabolized eATP to N⁶-etheno-ADP (eADP), eAMP and N⁶-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation in both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by sEV.

The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of the ecto-nucleotidase primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.