Purinergic activity of circulating extracellular vesicles associates with disease progression in melanoma

Extracellular vesicles (EVs) produced by melanoma cells (MTEX) carry ecto-nucleotidases, CD39/CD73, on their surface membrane, produce adenosine (ADO), and deliver it to immune recipient cells, inducing immune suppression. Based on studies of MTEX from melanoma cell lines, we hypothesized that MTEX are the major producers of ADO in cancer patients' plasma, which contains a variable mix of MTEX and NTEX. To test this hypothesis, EVs were isolated from the plasma of melanoma patients with no evidence of disease (NED) or with metastatic disease at phlebotomy, and MTEX were separated from EVs produced by non-malignant cells (NTEX) by immune capture using anti-CSPG4 mAbs. The purinergic activity of CD39/CD73 in total plasma EVs and NTEX was measured by high-pressure liquid chromatography with fluorescence detection, and that in MTEX was calculated. Unexpectedly, NTEX showed higher levels of purinergic activity than MTEX. While higher purinergic activity of NTEX vs. MTEX was not associated with disease status, the CD73 activity in total EVs was correlated with disease progression: it was significantly higher in patients with metastatic disease compared to those with NED (P = 0.02). Our data suggest that in the plasma of cancer patients, both MTEX and NTEX contribute to the purinergic metabolism mediated by EVs. Thus, the total EV fraction in plasma of melanoma patients provides a clinically meaningful assessment of purinergic activity and potentially can serve as a personalized estimate of levels of ADO-driven immune suppression in patients.