Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 1. CD73 gene expression is associated with poor PDAC prognosis. (A) Meta-analysis of CD73 gene expression (median) with 5-year overall survival (5-y OS). Forest plot displays the log hazard ratios (logHR) and 95% confidence intervals (CI). Horizontal bars represent the 95% CI of effect-size. Blue diamond represents the overall effect in all PDAC patients. (B) Association between CD73 gene expression (median) and OS in CD39-High PDAC (CD73 Low: n=37; CD73 High: n=36) and CD39-Low PDAC (CD73 Low: n=37; CD73 High: n=36) from the TCGA cohort. (C) Spearman correlation heatmap between ENTPD1 (CD39), NT5E (CD73), PDCD1 (PD-1), LAG3,

Article Snippet: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Gene Expression

Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 2. Tumor CD73 and stromal CD39 protein expression associate with poor PDAC prognosis and suppressed immune surveillance. (A-B) Representative immunofluorescence staining of CD73 expression in epithelium (A) and CD39 expression in stroma (B) of PDAC tumors. Epithelium was determined as the compartment expressing cytokeratin (CK, green). Stroma is the CK-negative compartment. CD73, red; DAPI, blue. (C-D) Mean fluorescence intensity (MFI) of epithelial and stromal CD73 (C) and CD39 (D) expression in 104 normal adjacent pancreas (N) versus intratumoral compartment (IT) (red bars indicate mean). (E-F) Association between CD73

Article Snippet: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 3. CD73 on tumor cells and myeloid cells promotes mouse PDAC. (A) CD73-positive (pos) or -negative (neg) KPC tumor cells were injected s.c. into C57BL/6 mice and treated with gemcitabine (gem; i.p. 100 mg/kg) on days 5 and 8. Mean tumor sizes are shown ± SEM (n=9–11). (B) KPC tumors were analyzed at day 11 by qPCR for expression of selected immune genes. Data represent mean relative expression ± SEM compared to CD73-positive tumors. (C) KPC tumors were analyzed at day 11 by FACS to assess the proportion of CD11b+Ly6G−Ly6Clo/− cells (TAMs) expressing CD206. Data represent individual and mean frequencies of TAM ± SEM gated on CD45+ cells. (D-E) CD73-negative KPC tumor cells were injected s.c. into CD73fl/fl LysMCre−/− mice (n=9) and into CD73fl/fl LysMCre+/− mice (n=12). On day 11, tumors were weighed (D) and the proportion of CD206+ TAMs was assessed by FACS. Data are representative of 2 independent experiments (n=9–10/group). Statistical comparisons were performed using one-way ANOVA comparing indicated groups (A, C), multiple t-test or unpaired t-test (B, D, E). *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Article Snippet: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Injection, Expressing

Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 4. CD39 cooperates with CD73 to promote mouse PDAC. (A) KPC tumor cells were injected s.c. into CD39fl/fl LysMCre−/− mice and CD39fl/fl

Article Snippet: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Injection

Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 5. CD73 promotes DNA damage repair and suppresses cGAS-STING activation. (A-B) CD73-positive (pos) or -negative (neg) KPC and PANC1 cells were exposed to increasing concentrations of gemcitabine, and proliferation was measured using CellTiter-Glo (Promega) after 48 hours of treatment. Data represents the average relative luminescence units (RLU) ± SEM of triplicates and is representative of 4 independent experiments. (C) Meta-analysis of CD73 gene association with sensitivity to gemcitabine in PDAC cell lines from the Genentech Cell Line Screening Initiative (gCSI), the Genomics of Drug Sensitivity in Cancer (GDSC), and the Cancer Therapeutics Response Portal (CTRP) datasets. Forest plot displaying the C-Index and 95% confidence intervals (CI) for each dataset. Horizontal bars represent the 95% confidence intervals of effect-size. The blue diamond represents the overall effect of the variable. (D) Representative images of γ-H2AX foci detected by immunofluorescence. (E) Number of γ-H2AX foci detected by immunofluorescence in CD73-positive or -negative KPC tumor cells following 48 hours of gemcitabine treatment at the indicated doses (n=4; mean ± SEM). (F) Number of γ-H2AX foci over time detected by immunofluorescence in KPC tumor cells following 1 Gy irradiation (n=3; mean ± SEM). (G) Number of γ-H2AX foci detected in human PANC1 cells treated with gemcitabine, CD73 inhibitor AB680, and/or A2B receptor agonist BAY 60–6583 (n=3; mean ± SEM). (H) Number of γ-H2AX foci detection after 48 hours of gemcitabine treatment in KPC tumor cells, in presence or absence of BAY 60–6583 (A2B receptor agonist)(n=3; mean ± SEM). (I) CD73-positive or -negative KPC tumor cells were exposed to gemcitabine (20 nM) for 48 hours and cGAMP was measured in supernatants by ELISA (n=3; mean ± SEM). (J) Following gemcitabine exposition, cGAS- induced gene expression in KPC cells was measured by qPCR (n=2; mean ± SEM). (K) CD73-positive KPC tumor cells expressing cGAS or not were injected in mice (n=10 per group). Some groups were treated with AB680 (peri-tumoral, 10mg/kg daily from day 6 to 11). Means ± SEM tumor growth at day 20 are shown (experiment performed once). Statistical significance was determined with Student T test (A-B, E-K). *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Article Snippet: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Activation Assay, Immunofluorescence, Irradiation, Enzyme-linked Immunosorbent Assay, Gene Expression, Expressing, Injection

Journal: Scientific Reports

Article Title: Role of P2X 4 Receptor in Mouse Voiding Function

doi: 10.1038/s41598-018-20216-4

Figure Lengend Snippet: Immunoprecipitation with anti-P2X 1 antibody. Antibodies to P2X 1 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads were resolved by SDS-PAGE, and Western blots were probed with A) P2X 1, B) P2X 4 or C) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. Monomeric P2X 1 can be seen highly concentrated in the pulldown fraction at 50 kDa. Little P2X 1 appears in FT. ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type, but is completely absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to possibly P2X 1 . Note, there is no evidence of the 70 kDa band in the IP lane. ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 1 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Article Snippet: The isolated proteins were resolved on 8–16% polyacrylamide gradient gels under reducing condition (+0.1 M DTT and 95 °C for 5 min before loading), and further blotted and probed with anti-P2X 1 , anti-P2X 4 and anti-5′-nucleotidase (NT5E) (Catalogue #: MAB44881, R&D system) antibodies for protein detection.

Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Protein Binding

Journal: Scientific Reports

Article Title: Role of P2X 4 Receptor in Mouse Voiding Function

doi: 10.1038/s41598-018-20216-4

Figure Lengend Snippet: Immunoprecipitation with anti-P2X 4 antibody. Antibodies to P2X 4 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads, were resolved by SDS-PAGE, and Western blots were probed with A ) P2X 1, B) P2X 4 or C ) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. P2X 1 is highly concentrated in the FT fractions. Minor potential P2X 1 staining appears in the IP lane, however this is due to P2X 4 antibody cross-reacting and pulling down some P2X 1 . ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type IP lane, but is absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to P2X 1 (50 kDa band). ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 4 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Article Snippet: The isolated proteins were resolved on 8–16% polyacrylamide gradient gels under reducing condition (+0.1 M DTT and 95 °C for 5 min before loading), and further blotted and probed with anti-P2X 1 , anti-P2X 4 and anti-5′-nucleotidase (NT5E) (Catalogue #: MAB44881, R&D system) antibodies for protein detection.

Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Staining, Protein Binding

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: When N6-etheno-ATP (a) and N6-etheno-AMP (b) were incubated for 30 min at 30 °C in the absence of ecto-nucleotidases, the only chromatographic peaks observed were intact N6-etheno-ATP and intact N6-etheno-AMP, respectively, thus indicating that N6-etheno-ATP and N6-etheno-AMP were chemically stable under these test conditions. When N6-etheno-ATP was incubated for 30 min at 30 °C in the presence of either 20 ng of rhCD39 (c), 80 ng of rhENPP-1 (d), 40 ng of rhENTPD2 (e), or 11 ng of rhENTPD3 (f), N6-etheno-ATP was essentially quantitatively converted to N6-etheno-AMP. When N6-etheno-AMP (g) was incubated for 30 min at 30 °C in the presence of rhCD73 (40 ng), all of the N6-etheno-AMP was recovered as N6-etheno-adenosine (ADO)

Article Snippet: Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: Scatter plots show the percentage (%) of applied substrate (either the natural adenine nucleotide substrate or the corresponding etheno-bridged adenine nucleotide substrate, both at 1 μmol/L) that remained or was recovered as product (either the natural product or corresponding etheno-bridged product) after incubation (5 min at 30 °C) with recombinant human (rh) ecto-nucleotidases a rhENPP-1, b rhENTPD2, c rhENTPD3, d rhCD73, or e rhCD39. For each ecto-nucleotidase, the amount of enzyme incubated with substrate was selected to only partially metabolize the natural adenine nucleotide substrate. eATP = N6-etheno-ATP; eADP = N6-etheno-ADP; eAMP = N6-etheno-AMP; eADO = N6-etheno-adenosine (eADO). *P < 0.05 versus corresponding natural substrate. All individual data points are provided along with the means and SDs

Article Snippet: Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation, Recombinant

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: To determine initial reaction velocities, CD39 (10 ng) was incubated with high concentrations of substrates (25 to 200 μmol/L) for 10 min at 30 °C. In panel a, substrates were either ATP or N6-etheno-ATP and the downstream products (ADP + AMP or N6-etheno-ADP + N6-etheno-AMP) were measured. In panel b, substrates were either ADP or N6-etheno-ADP and the downstream products (AMP or N6-etheno-AMP) were measured. The experiment in panel c was similar to that described for panel b with the exception that the substrates were AMP or N6-etheno-AMP, the enzyme was CD73 (0.25 ng), and the measured products were adenosine and N6-etheno-ADO. eATP = N6-etheno-ATP; eADP = N6-etheno-ADP; eAMP = N6-etheno-AMP. Values represent means ± SDs

Article Snippet: Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: A CT26 CRC cells were stained with PKH26GL (red) and quinacrine (green) fluorescent dyes. Images were acquired using confocal microscopy at time 0 and after 5 min following P2X7 activation with 300 µM BzATP and are extrapolated from a 30-min time course (see supplementary videos and ). B CT26 cells were pre-treated with P2X7 antagonist AZ10606120 (5 µM) for 10 min before application of BzATP. C Number of vesicles released in 30 min from CT26 cells in PBS vehicle (PBS S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist AZ10606120 followed by stimulation with 3 mM ATP (AZ-VS) ( n = 5). D Size of PBS S-VS, P2X7-VS, and AZ-VS ( n = 5). E Number of vesicles released in 30 min from CT26 cells in DMSO vehicle (DMSO S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist A740003 followed by stimulation with 3 mM ATP (A74-VS) ( n = 7). F Size of DMSO- S-VS, P2X7-VS, and A74-VS ( n = 7). G Western blot for GM130, Alix, P2X7, CD39, CD73, and A2A in CT26 cells, S-VS and P2X7-VS. Pericellular ATP was measured with the pmeLUC probe expressed on the cell surface of untreated CT26 cells or after 5 min of exposure to PBS vehicle, S-VS, and P2X7-VS ( n = 4). H Quantification of luminescence changes was expressed as a fold increase on time 0. I Representative images of photon emissions. Changes in ATP J concentration increase on time 0 in the supernatants of CT26 cells, untreated or treated with PBS vehicle, S-VS, or P2X7-VS, measured with a luciferin/luciferase assay ( n = 3). Changes in adenosine K concentration increase on time 0 in the supernatants of CT26 cells untreated or treated with PBS vehicle, S-VS, P2X7-VS, or P2X7-VS plus 5uM CD73 inhibitor AB680 ( n = 5). * p < 0.05, ** p < 0.001, *** p < 0,0001, **** p < 0.00001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Staining, Confocal Microscopy, Activation Assay, Western Blot, Concentration Assay, Luciferase

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: The mRNA expression of P2X7A A , B , P2X7B C , D , CD39 E , F , CD73 G , H , and A2A I , J was evaluated in the cDNAs of 158 patients with CRC subdivided into stage I ( n = 24), stage II ( n = 50), stage III ( n = 52), and stage IV ( n = 32) which comprised 11 samples derived from metastases in organs other than the colon. K Spearman’s correlation coefficient among P2X7A, P2X7B, CD39, CD73 , and A2A was evaluated in CRC metastatic patients. L Spearman’s correlation coefficient was evaluated between P2X7 and A2A expression in colon adenocarcinoma samples obtained from the Cancer Genome Atlas database. * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Expressing, Derivative Assay

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: mRNA expression of A P2X7A , B P2X7B , C A2A , D CD39 , and E CD73 in CRC patients subdivided into APC WT and APC mutated groups ( n = 6). Percentage of cells positive for P2X7 F and A2A G in the colons of WT and PIRC rats and PIRC tumors ( n = 4). Representative images of immunohistochemical staining for P2X7 and A2A in the colon of WT 1-year rats H, K and in the normal colon I, L and the tumor mass J, M of 1-year PIRC rats. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining