Journal: bioRxiv

Article Title: Breast cancer extracellular vesicles transfer P2X7 signaling competence to endothelial cells and dynamically remodel vascular migration

doi: 10.64898/2026.07.31.741755

Figure Lengend Snippet: a) NTA profile of representative EVs batches isolated from MCF-7- (left graph) and PANC-1- (right graph) conditioned medium. The MCF-7-derived batch displayed a predominant peak at 138 nm (mode), with a mean particle size of 209 nm and a concentration of 1.58 x 10 11 particles/mL. The PANC-1-derived batch displayed a predominant peak at 137 nm (mode), with a mean particle size of 180 nm and a concentration of 8.8 x 10 10 particles/mL. b) Representative TEM micrographs of negatively stained SEC-purified EVs isolated from MCF-7 (left panels) and PANC- 1 (right panels) conditioned media. Lower panels show higher-magnification views of the boxed regions indicated in the corresponding upper panels. Scale bars: 1 μm (upper panels) and 250 nm (lower panels). c) Quantification of wound healing closure by HMEC-1 after 72-hour treatment with CM from MCF-7 (left graph) or PANC-1 (right graph), EV-deprived CMs, and EVs isolated from the CMs. Data are expressed as % wound closure after 16-hour treatment with 100 μM BzATP relative to time = 0 h and represented as mean ± SEM of at least 4 independent experiments (n≥4). Statistical significance: n.s., not significant = p > 0.05; * = p < 0.05; *** = p < 0.001 (paired t-test or Wilcoxon test according to data distribution). d) Schematic representation and quantification of the P2X7-dependent % of cell migration inhibition following direct co-culture with MCF-7 for 72 h in Transwell systems, 72-hour treatment with CM-MCF-7 in a similar ratio of MCF-7 medium/HMEC-1 medium to that in Transwell systems, and 72-hour treatment with EVs isolated from CM-MCF-7 and used at the same concentration as in CM. Statistical significance: n.s., not significant = p > 0.05; **** = p < 0.0001 (unpaired t-test for w/MCF-7 vs CM-MCF-7 comparison, and paired t-test for CM-MCF-7 vs EV-MCF-7 comparison).

Article Snippet: Treatments were then applied, including stimulation with BzATP (MedChemExpress, Cat. #HY-136254, 100 μM) alone or in combination with a non-selective P2X receptor antagonist (PPADS, Sigma-Aldrich Cat. #P178, 100 μM) or a selective P2X7 antagonist (A438079, MedChemExpress, Cat. #HY-15488A, 10 μM), as well as antagonist-only conditions.

Techniques: Isolation, Derivative Assay, Concentration Assay, Staining, Purification, Migration, Inhibition, Co-Culture Assay, Comparison

Journal: bioRxiv

Article Title: Breast cancer extracellular vesicles transfer P2X7 signaling competence to endothelial cells and dynamically remodel vascular migration

doi: 10.64898/2026.07.31.741755

Figure Lengend Snippet: a) Representative images (magnification: 4X; scale bar: 200 μm) and quantification of wound healing closure by HMEC-1 co-cultured in Transwell systems for 72 h with themselves (HMEC-1 w/HMEC-1), MCF-7 (HMEC-1 w/MCF-7), PANC-1 (HMEC-1 w/PANC-1), and PC3 (HMEC-1 w/PC3) after 16 and 24 h of treatment with 100 μM BzATP. Data are expressed as % wound closure relative to time = 0 h and represented as mean ± SEM of 5 independent experiments (n=5). Statistical significance: n.s., not significant = p > 0.05; *** = p < 0.001 (paired t-test or Wilcoxon test according to data distribution) b) Cell viability assays on HMEC-1 co-cultured with themselves (HMEC-1 w/HMEC-1), or with MCF-7 (HMEC-1 w/MCF-7) after 24 h of treatment with 100 μM BzATP. Data are normalized on the control (HMEC-1 w/HMEC-1 untreated) and are represented as mean ± SEM. Data refer to the mean of four independent experiments (n=4). Statistical significance: n.s., not significant = p > 0.05 (paired t-test). c) Quantification of wound healing closure by HMEC-1 w/MCF-7 after 16 and 24 h of treatment with 100 μM BzATP in combination or without 100 μM PPADS (left graph) or 10 μM A438079 (right graph). Inhibitors were also tested alone as a control. Data are expressed as % wound closure relative to time = 0 h and represented as mean ± SEM of 5 independent experiments (n=5). Statistical significance: n.s., not significant = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (paired t-test or Wilcoxon test according to data distribution).

Article Snippet: Treatments were then applied, including stimulation with BzATP (MedChemExpress, Cat. #HY-136254, 100 μM) alone or in combination with a non-selective P2X receptor antagonist (PPADS, Sigma-Aldrich Cat. #P178, 100 μM) or a selective P2X7 antagonist (A438079, MedChemExpress, Cat. #HY-15488A, 10 μM), as well as antagonist-only conditions.

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Breast cancer extracellular vesicles transfer P2X7 signaling competence to endothelial cells and dynamically remodel vascular migration

doi: 10.64898/2026.07.31.741755

Figure Lengend Snippet: a) Representative Ca 2+ -imaging traces in response to P2X7 agonist (100 μM BzATP) in HMEC-1 co-cultured with themselves (grey trace), or MCF-7 (pink trace). Traces represent the mean ± SEM of cells in the recorded field of one representative experiment (n = 14 for HMEC-1 w/HMEC-1, n = 12 for HMEC-1 w/MCF-7). The comparison between HMEC-1 w/HMEC-1 and HMEC-1 w/MCF-7 in terms of basal Ca 2+ content is reported on the right (n.s.: not significant = p > 0.05; paired t-test). Quantification of the BzATP- (b) and ATP- (c) induced intracellular Ca 2+ responses in terms of % of responsive cells, peak amplitude, and area of the signals in responsive cells. Data represent the mean ± SEM of 8 independent experiments (n=8). Statistical significance: * = p < 0.05; ** = p < 0.01 (paired t-test or Wilcoxon test according to data distribution).

Article Snippet: Treatments were then applied, including stimulation with BzATP (MedChemExpress, Cat. #HY-136254, 100 μM) alone or in combination with a non-selective P2X receptor antagonist (PPADS, Sigma-Aldrich Cat. #P178, 100 μM) or a selective P2X7 antagonist (A438079, MedChemExpress, Cat. #HY-15488A, 10 μM), as well as antagonist-only conditions.

Techniques: Imaging, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Breast cancer extracellular vesicles transfer P2X7 signaling competence to endothelial cells and dynamically remodel vascular migration

doi: 10.64898/2026.07.31.741755

Figure Lengend Snippet: a) Quantification of wound healing closure by HMEC-1 following treatment for 72h with conditioned medium (CM) from HMEC-1 (CM-HMEC-1), MCF-7 (CM-MCF-7), or PANC-1 (CM-PANC-1). CMs were used at a similar ratio of cancer cells medium/HMEC-1 medium to that in Transwell systems (1:3). Data are expressed as % wound closure after 16-hour treatment with 100 μM BzATP relative to time = 0h and represented as mean ± SEM of at least 4 independent experiments (n≥4). The effect of 10 μM A438079 alone and in combination with 100 μM BzATP was evaluated to confirm the specificity of P2X7 in the inhibition observed in HMEC-1 treated with MCF-7-conditioned medium (CM-MCF-7). Statistical significance: n.s., not significant = p > 0.05; * = p < 0.05 (paired t-test or Wilcoxon test according to data distribution). b) Quantification of wound healing closure by HMEC-1 following treatment for 72h with increasing concentration of conditioned medium from MCF-7 (dilution 1:4, 1:3, and 1:2). Data are expressed as % wound closure 16h after treatment with 100 μM BzATP relative to time = 0h and represented as mean ± SEM of 6 independent experiments (n=6). Statistical significance: n.s., not significant = p > 0.05; * = p < 0.05 (Wilcoxon test).

Article Snippet: Treatments were then applied, including stimulation with BzATP (MedChemExpress, Cat. #HY-136254, 100 μM) alone or in combination with a non-selective P2X receptor antagonist (PPADS, Sigma-Aldrich Cat. #P178, 100 μM) or a selective P2X7 antagonist (A438079, MedChemExpress, Cat. #HY-15488A, 10 μM), as well as antagonist-only conditions.

Techniques: Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Breast cancer extracellular vesicles transfer P2X7 signaling competence to endothelial cells and dynamically remodel vascular migration

doi: 10.64898/2026.07.31.741755

Figure Lengend Snippet: Quantification of wound healing closure by HMEC-1 at increasing times from the interruption of co-culture with MCF-7 in Transwell systems (from 1 to 4 days). Data are expressed as % wound closure after 16-hour treatment with 100 μM BzATP relative to time = 0h and represented as mean ± SEM of at least 4 independent experiments (n≥4). The percentage of P2X7-dependent inhibition of cell migration is reported for each time point. Statistical significance: * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (paired t-test).

Article Snippet: Treatments were then applied, including stimulation with BzATP (MedChemExpress, Cat. #HY-136254, 100 μM) alone or in combination with a non-selective P2X receptor antagonist (PPADS, Sigma-Aldrich Cat. #P178, 100 μM) or a selective P2X7 antagonist (A438079, MedChemExpress, Cat. #HY-15488A, 10 μM), as well as antagonist-only conditions.

Techniques: Co-Culture Assay, Inhibition, Migration

Journal: Frontiers in Aging Neuroscience

Article Title: 40-Hz transauricular vagal nerve stimulation rescues cognition of 9-month-old APP/PS1 mice via inhibiting hippocampal P2X7 receptor signaling

doi: 10.3389/fnagi.2026.1766813

Figure Lengend Snippet: Illustration of experimental groups, parts, and timeline. There are 11 groups in total: (1) WT, (2) WT + 40 Hz-taVNS, (3) APP/PS1, (4) APP/PS1 + 40-Hz taVNS, (5) APP/PS1 + 8-Hz taVNS, (6) APP/PS1 + 80-Hz taVNS, (7) APP/PS1 + vehicle-01, (8) APP/PS1 + A-804598, (9) APP/PS1 + vehicle-02, (10) APP/PS1 + BzATP, and (11) APP/PS1 + BzATP + 40-Hz taVNS groups. The work includes 3 parts. Part 1 includes (1) WT, (2) WT + 40 Hz-taVNS, (3) APP/PS1, (4) APP/PS1 + 40-Hz taVNS groups; Part 2 includes (3) APP/PS1, (4) APP/PS1 + 40-Hz taVNS, (5) APP/PS1 + 8-Hz taVNS, (6) APP/PS1 + 80-Hz taVNS groups; Part 3 includes (3) APP/PS1, (4) APP/PS1 + 40-Hz taVNS, (7) APP/PS1 + vehicle-01, (8) APP/PS1 + A-804598, (9) APP/PS1 + vehicle-02, (10) APP/PS1 + BzATP, and (11) APP/PS1 + BzATP + 40-Hz taVNS groups. Timeline: Time points for daily (injection) stimulation; habituation phase of novel object recognition (NOR) and novel object location (NOL); training and testing of NOR; training and testing of NOL; acquisition training and probe trial of Morris water maze (MWM); and tissue harvest. Daily (injection) stimulation (taVNS at 8 Hz, 40 Hz, or 80 Hz) began on day 1 when the mice reached 9 months old and continued for a duration of 16 days. The (injection) stimulation was administered in the morning (8:00–12:00 a.m.), and behavioral tests were conducted in the afternoon (2:00–5:00 p.m.). On day 16, tissue was harvested in the afternoon (2:00–5:00 p.m.).

Article Snippet: A-804598 (a P2X7 receptor antagonist, 30 mg/kg/day, Ambeed, A270115) ( ) and BzATP (a P2X7 receptor agonist, 30 nmol in 1 μL saline, Ambeed, A433956) ( ) were used in this investigation.

Techniques: Injection

Journal: Frontiers in Aging Neuroscience

Article Title: 40-Hz transauricular vagal nerve stimulation rescues cognition of 9-month-old APP/PS1 mice via inhibiting hippocampal P2X7 receptor signaling

doi: 10.3389/fnagi.2026.1766813

Figure Lengend Snippet: The results of the NOR, NOL, and MWM tests in part 3 of the study. (a) The recognition index of the NOR test ( n = 9 per group, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, * p < 0.05; APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, # p < 0.05; APP/PS1 vs. APP/PS1 + vehicle-01 and APP/PS1 vs. APP/PS1 + vehicle-02, n.s., not significant, p > 0.05). (b) The average velocity (cm/s), during the NOR test ( n = 9 per group, APP/PS1 vs. APP/PS1 + vehicle-01, APP/PS1 vs. APP/PS1 + vehicle-02, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, n.s., not significant, p > 0.05). (c) The cumulative distance (cm) traveled throughout the NOR test ( n = 9 per group, APP/PS1 vs. APP/PS1 + vehicle-01, APP/PS1 vs. APP/PS1 + vehicle-02, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, n.s., not significant, p > 0.05). (d) The recognition index of the NOL test ( n = 9 per group, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, * p < 0.05; APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, # p < 0.05; APP/PS1 vs. APP/PS1 + vehicle-01 and APP/PS1 vs. APP/PS1 + vehicle-02, n.s., not significant, p > 0.05). (e) The average velocity (cm/s), during the NOL test ( n = 9 per group, APP/PS1 vs. APP/PS1 + vehicle-01, APP/PS1 vs. APP/PS1 + vehicle-02, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, n.s., not significant, p > 0.05). (f) The cumulative distance (cm) traveled throughout the NOL test ( n = 9 per group, APP/PS1 vs. APP/PS1 + vehicle-01, APP/PS1 vs. APP/PS1 + vehicle-02, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, n.s., not significant, p > 0.05). (g) Escape latencies in the MWM ( n = 9 per group, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, * p < 0.05; APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, # p < 0.05). (h) Time spent swimming in the goal quadrant during the probe trial ( n = 9 per group, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, * p < 0.05; APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, # p < 0.05; APP/PS1 vs. APP/PS1 + vehicle-01 and APP/PS1 vs. APP/PS1 + vehicle-02, n.s., not significant, p > 0.05). (i) Number of platform crossings during the probe trial ( n = 9 per group, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, * p < 0.05; APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, # p < 0.05; APP/PS1 vs. APP/PS1 + vehicle-01 and APP/PS1 vs. APP/PS1 + vehicle-02, n.s., not significant, p > 0.05). (j) Average swimming velocity (cm/s) during MWM ( n = 9 per group, APP/PS1 vs. APP/PS1 + vehicle-01, APP/PS1 vs. APP/PS1 + vehicle-02, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, n.s., not significant, p > 0.05).

Article Snippet: A-804598 (a P2X7 receptor antagonist, 30 mg/kg/day, Ambeed, A270115) ( ) and BzATP (a P2X7 receptor agonist, 30 nmol in 1 μL saline, Ambeed, A433956) ( ) were used in this investigation.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: 40-Hz transauricular vagal nerve stimulation rescues cognition of 9-month-old APP/PS1 mice via inhibiting hippocampal P2X7 receptor signaling

doi: 10.3389/fnagi.2026.1766813

Figure Lengend Snippet: Hippocampal Aβ42, P2X7R, NLRP3, Caspase-1 expression, and Aβ1-40, Aβ1-42, NF-κB, pro-IL-1β, pro-IL-18, IL-1β, IL-18 levels in part 3 of the study. WB results show hippocampal Aβ42 (a) , P2X7R (b) , NLRP3 (c) , and Caspase-1 (20 kDa) (d) expression ( n = 6 per group, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, * p < 0.05; APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, # p < 0.05; APP/PS1 vs. APP/PS1 + vehicle-01 and APP/PS1 vs. APP/PS1 + vehicle-02, n.s., not significant, p > 0.05. ELISA results show soluble Aβ1-40 (e) , soluble Aβ1-42 (f) , NF-κB (g) , pro-IL-1β (h) , pro-IL-18 (i) , IL-1β (j) , and IL-18 (k) levels in the hippocampi of the mice. n = 6 per group, APP/PS1 vs. APP/PS1 + 40-Hz taVNS, APP/PS1 + vehicle-01 vs. APP/PS1 + A-804598, and APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP + 40-Hz taVNS, * p < 0.05; APP/PS1 + vehicle-02 vs. APP/PS1 + BzATP, # p < 0.05; APP/PS1 vs. APP/PS1 + vehicle-01, and APP/PS1 vs. APP/PS1 + vehicle-02, n.s., not significant, p > 0.05).

Article Snippet: A-804598 (a P2X7 receptor antagonist, 30 mg/kg/day, Ambeed, A270115) ( ) and BzATP (a P2X7 receptor agonist, 30 nmol in 1 μL saline, Ambeed, A433956) ( ) were used in this investigation.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay