Fig 2: Graphical summary of results. Upon agonist-induced platelet activation, ATP and ADP are released. These are converted to AMP by platelet CD39 activity. AMP is converted to adenosine by MSC-expressed CD73 and to a low extent by alkaline phosphatase. Adenosine signals vial A2AR and other P1 receptors to raise cAMP levels and to induce VASP phosphorylation. This reduces further platelet activation. Used inhibitors indicated in red. ADA adenosine deaminase, ADP adenosine diphosphate, ALP alkaline phosphatase, AMP adenosine monophosphate, ATP adenosine triphosphate, TRAP thrombin receptor activator for peptide, VASP vasodilator-stimulated phosphoprotein

Fig 3: Alkaline phosphatase and adenosine deaminase activity and function blocking. a Alkaline phosphatase (ALP) and b adenosine deaminase (ADA) activity in different cell types. Individual biological replicates depicted as dots. c Effects induced by adding ALP inhibitor levamisole or ADA in platelet–cell cocultures. Data normalized against control activated by TRAP-6 without cells (dotted line at value 1). w/o n = 4–11, BM-MSCs n = 9, LA-MSCs n = 7–12, CB-MSCs n = 5–9, HUVECs n = 6 biological replicates; HeLa cells n = 3, adenosine n = 4. **p < 0.01. BM bone marrow, CB cord blood, HUVEC human umbilical vein endothelial cell, LA lipoaspirate, MFI mean fluorescence intensity, w/o without

Fig 4: RSV treatment effect on adenosine-related enzymatic activities. 5′-nucleotidase (CD73) and ADA activities were measured in control and 24 h RSV-treated C6 cells. (A) 5′-nucleotidase activity localized in the plasma membrane and cytosolic fraction were assayed and represented as nmol Pi/mg prot ⋅ min. (B) ADA activity was quantified and represented as pmol/μg prot ⋅ min. (C) Cell viability based on the XTT method was performed after 24 h of treatment with the indicated ligands. (D) Quantification of adenosine levels in culture medium by HPLC. Data are means ± SEM of three to five independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 significantly different from the control condition according to Student’s t-test.

Fig 5: Possible role of adenosinergic signaling in the antitumoral effect of RSV. The exposure of C6 glioma cells to RSV causes the decrease (↓) or the increase (↑) of several compounds and proteins that ultimately lead to cell cycle arrest in the G1 phase. RSV upregulates A1 and A3 receptors; changes G-protein coupling of A2A receptor from activation (Gs, +) to inhibition (Gi, −) of adenylate cyclase (AC) activity; upregulates AC, increases basal AC activity, and upregulates protein kinase A (PKA); higher levels of cAMP inhibit AMPK activation via PKA-mediated phosphorylation (Ser172); reduces adenosine-converting enzymes (i.e., CD73 and ADA), leading to increased extracellular levels of adenosine; caspase-3 is activated as a result of the direct binding of RSV to adenosine receptors and the modulatory action of these receptors on other RSV-elicited pathways, leading to CDK-mediated cell cycle arrest in the G1 phase. Previous results reported by our group in these cells are shown in gray and surrounded by a dotted line.