Fig 2: C57bl/6 mice were inoculated into the right flank with B16-F10 cells in wild type and P2X7 null mice. A2AR antagonist SCH58261 (1 mg/kg) or placebo (sterile PBS containing 0.005% DMSO) were intra-peritoneum injected every 3 days after first tumor mass detection (day 5 from inoculum). (A) Ex-vivo tumor volume assessed by calliper. (B) Representative pictures of tumors derived from treated wild type and P2X7 null mice at post inoculum day 14 (n = 7–11 per group). (C–F) A2AR (C) and P2X7R (E) expression by Western Blot and relative quantification (D,F) (n = 5–13 per group). VEGF (G) and TGF-ß (H) were evaluated in tumor masses obtained at post inoculum day 14 (n = 3–6 per group). Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig 3: A2AR do not affect spontaneous or evoked excitatory synaptic transmission in the mPFC. Biocytin staining of layer 5 pyramidal neuron (A, top) and fast spiking (FS) interneuron (H, top) from coronal slice of rat prelimbic mPFC. Scale bars: 100 µm. Voltage responses to hyperpolarizing (–50 pA) and depolarizing (+225 pA) somatic current injections to the soma of a L5 pyramidal neuron (A, bottom) and to hyperpolarizing (–60 pA) and depolarizing (+400 pA) somatic current injections to the soma of a FS interneuron (H, bottom). Scale bars: 20 mV, 200 ms. (B,C) Bath application of the selective A2AR antagonist, SCH58261 (100 nM, blue) did not affect the frequency of sEPSCs onto pyramidal neurons. A representative trace is depicted in control (black) and prolonged SCH58261 (100 nM, blue) wash-in condition (B). The frequency over 5 min does not change after SCH58261 (100 nM, blue) wash-in; data are mean ± SD of n = 10, individual lines show the average of one cell over the course of 5 min per condition (C). Scale bars: 30 pA, 50 ms. (D,E) Bath application of SCH58261 (100 nM, blue) did not affect the amplitude of sEPSCs onto pyramidal neurons. The average sEPSC amplitude of a representative cell is depicted in control (black) and prolonged SCH58261 (100 nM, blue) wash-in condition (D). The average amplitude over 5 min does not change after SCH58261 (100 nM, blue) wash-in; data are mean ± SD of n = 10 (E). Scale bars: 10 pA, 1 ms. (F,G) Bath application of SCH58261 (100 nM, blue) did not affect the amplitude of eEPSCs onto pyramidal neurons. The eEPSC amplitude of a representative cell is depicted during 15 sweeps in all three conditions; control (black), short- (light blue) and prolonged SCH58261 (100 nM, blue) wash-in conditions are shown (F). The average amplitude does not change after SCH58261 (100 nM, blue) wash-in; data are mean ± SD of n = 14 (G). (I,J) Bath application of SCH58261 (100 nM, blue) did not affect the frequency of sEPSCs onto interneurons. A representative trace is depicted in control (black) and prolonged SCH58261 (100 nM, blue) wash-in condition (I). The frequency over 5 min does not change after SCH58261 (100 nM, blue) wash-in; data are mean ± SD of n = 12, individual lines show the average of one cell over the course of 5 min per condition (J). Scale bars: 30 pA, 50 ms. (K,L) Bath application of SCH58261 (100 nM, blue) did not affect the amplitude of sEPSCs onto interneurons. The average sEPSC amplitude of a representative cell is depicted in control (black) and prolonged SCH58261 (100 nM, blue) wash-in condition (K). The average amplitude over 5 min does not change after SCH58261 (100 nM, blue) wash-in; data are mean ± SD of n = 12 (L). Scale bars: 10 pA, 1 ms. (M,N) Bath application of SCH58261 (100 nM, blue) did not affect the amplitude of eEPSCs onto pyramidal neurons. The average eEPSC amplitude of a representative cell is depicted over a time-course of 30 min, in which control (black), short- and prolonged SCH58261 (100 nM, blue) wash-in conditions are shown (M). The average amplitude over 5 min does not change after SCH58261 (100 nM, blue) wash-in; data are mean ± SD of n = 8 (N). Paired one-way ANOVA; all data are non-significant (p > 0.05).

Fig 4: A2AR facilitate LTP of population spikes in mPFC layer 5. (A) Positioning of the stimulating (layer 2/3 mPFC) and recording electrodes (layer 5 mPFC). (B) The recorded population spikes were abolished by the AMPA/kainate receptor antagonist, CNQX 10 μM. (C) Bath application of the selective A2AR antagonist, SCH58261 (50 nM; blue dots) increased the number and synchrony of cells discharging action potentials as indicated by the increase in the maximum response when the input/output response was assessed 20 min after SCH58261 superfusion. (D) Representative averaged traces at baseline and 30 min after the induction of LTP, in the absence and presence of SCH58261 (50 nM); SCH58261 decreased LTP magnitude. (E) Time course showing that SCH58261 decreased the magnitude of LTP of the population spike responses triggered by a priming train of high-frequency stimulation (HFS), followed 15 min later by four HFS trains (50 pulses at 100 Hz, 0.5 s duration, delivered every 10 s). (F) Summary plot displaying the variability of LTP magnitude, estimated 30 min after the induction of LTP, in the absence and presence of SCH58261 (50 nM). Data are mean ± SEM of 25 slices (from 25 rats) per group. ####p < 0.0001 comparing the estimated maximum response in the presence of SCH58261 to control (black bar/dots), unpaired Student’s t-test. ∗p < 0.05 comparing SCH58261 to control (black bar/dots), unpaired Student’s t-test. ∗∗∗∗p < 0.0001 compared to the hypothetical value of 100%, one-sample t-test.

Fig 5: (A,B) Immunohistochemistry of A2AR in tumors derived from wild type (A) and P2X7 null (B) C57bl/6 mice. Pictures are obtained with a 20X objective. (C) Intra-tumor levels of VEGF (wild type n = 5, P2X7 null n = 4). (D–F) Immunohistochemistry (D,E) and relative quantification (F) of CD31 in tumor derived from wild type and P2X7 null C57bl/6 mice (n = 4 per group). Data are shown as the mean ± SEM. *p < 0.05 and **p < 0.01.