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: 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: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. Structures of FAD and metabolites of FAD B. FMN and FAD hydrolysis by human bone alkaline phosphatase (ALP) and recombinant human CD73 Riboflavin (RF) and FMN concentrations were measured 15 min after incubation of 10 μM FMN or FAD, respectively, with ALP (10 μg/mL) or CD73 (10 ng/mL). Data represent means ± S.D. ( n = 3). N.D.: Not detectable.

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Recombinant, Incubation

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. Time course of RF production from FMN by ALP. The RF concentration was measured after incubation of 10 μM FMN with ALP (10 μg/mL). Data represent means ± S.D. ( n = 3). B. ALP concentration-dependence of RF production from FMN. The RF concentration was measured 5 min after incubation of 10 μM FMN with ALP (2.5–10 μg/mL). Data represent means ± S.D. ( n = 3). C. Michaelis-Menten plot of RF production from FMN by ALP. The RF concentration was measured 5 min after incubation of FMN (0.5–5 μM) with ALP (5 μg/mL). Data represent means ± S.D. ( n = 3). D, G. Time course of FMN production (D) or adenosine production (G) from FAD by CD73. The FMN or adenosine concentration was measured after incubation of 10 μM FAD with CD73 (10 ng/mL). Data represent means ± S.D. ( n = 3). E, H. CD73 concentration-dependence of FMN production (E) or adenosine production (H) from FAD by CD73. The FMN or adenosine concentration was measured 15 min after incubation of 10 μM FAD with CD73 (5–20 ng/mL). Data represent means ± S.D. ( n = 3). F, I. Michaelis-Menten plot of FMN production (F) or adenosine production (I) from FAD by CD73. The FMN or adenosine concentration was measured 15 min after incubation of 10 μM FAD with CD73 (10 ng/mL). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown.

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Concentration Assay, Incubation

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. Comparison of inhibitory effect of various compounds on hydrolysis of 1 μM FMN and 1 μM AMP by ALP. FMN hydrolysis activities were measured by RF production from FMN; AMP hydrolysis activities were measured by adenosine production from AMP. The concentration of the inhibitors was 10 μM, except for α,β-methylene adenosine 5′-diphosphate (APCP; 2 μM) and levamisole (1 mM). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown. B. Comparison of inhibitory effects of various compounds on hydrolysis of 10 μM FAD and 4 μM AMP by CD73. FAD hydrolysis activities were measured by FMN production from FAD; AMP hydrolysis activities were measured by adenosine production from AMP. The concentration of the inhibitors was 10 μM, except for APCP (2 μM) and levamisole (1 mM). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown. C. Comparison of inhibitory effects of various compounds on hydrolysis of 1 and 4 μM AMP by ALP and CD73, respectively. AMP hydrolysis activities were measured by adenosine production from AMP. The concentration of the inhibitors was 10 μM, except for APCP (2 μM) and levamisole (1 mM). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown.

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. ALP activities in phosphatidylinositol glycan anchor biosynthesis class T (PIGT)-expressing (PIGT+) or vector-transfected (PIGT−) SH-SY5Y cells. ALP activities were measured in lysates of PIGT+ and PIGT− cells. The ALP activity is expressed in terms of the amount of placental ALP (PLAP) in the kit, which was used as a positive control. Data represent means ± S.D. ( n = 4). ** Indicates a significant difference ( p < 0.01) compared with PIGT+ cells. B. CD73 activities in PIGT+ or PIGT− SH-SY5Y cells. CD73 activities were measured by APCP-sensitive adenosine production from AMP in lysate of PIGT+ or PIGT− SH-SY5Y cells. Data represent means ± S.D. ( n = 3). ** Indicates a significant difference ( p < 0.01) compared with PIGT+ cells. C. Surface expression of CD73, ALP, and CD59 on PIGT− and PIGT+ SHSY5Y cells. MFI (mean fluorescent intensity) of PIGT+ vs PIGT-; CD73, 177 vs 82; ALP, 472 vs 102; CD59, 10093 vs 77) The analysis was repeated at least three times. D. Residual amount of vitamin B 2 in medium after 24 h of cultivation of PIGT+ or PIGT− SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The residual amount of vitamin B 2 in medium is expressed as the percentage of the total vitamin B 2 concentration (FAD + FMN + RF) in medium incubated without cells. The data represent means for FAD (white), FMN (gray), and RF (black). Error bars represent the S.D. for total vitamin B 2 . ** Indicates a significant difference ( p < 0.01) in the residual amount of total vitamin B 2 in the medium. ND: Not detectable. E. Intracellular vitamin B 2 amount after 24 h of cultivation of PIGT+ or PIGT− SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The data represent means for FAD (white), FMN (gray), and RF (black). Error bars represent the S.D. for total vitamin B 2 . ** Indicates a significant difference ( p < 0.01) in the total vitamin B 2 concentration in cells. #, ## indicates a significant difference ( p < 0.05, p < 0.01, respectively) in FMN concentration in cells. †† indicates a significant difference ( p < 0.01) in FAD concentration in cells. ND: Not detectable. F. PLP and PL concentration in medium after 24 h of cultivation of PIGT+ or PIGT− SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The data represent means for PLP (gray) and PL (black). Error bars represent the S.D. of the sum of PLP and PL. ** Indicates a significant difference ( p < 0.01) in the sum of PLP and PL in the medium. ND: Not detectable. G. Correlation between intracellular FMN concentration and the sum of PL and PLP concentration after 24 h of cultivation of PIGT+ (closed circles) or PIGT− (open circles) SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The data represent means ± S.D. ( n = 3). Statistical analysis: Student’s t-test for (A, B); ANOVA followed by the Tukey–Kramer test for (D, E and F); Pearson correlation for (G).

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Expressing, Plasmid Preparation, Transfection, Activity Assay, Positive Control, Concentration Assay, Incubation

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A, B. Cells were incubated in the measuring plates with vitamin B 2 -depleted medium for 4 days. The medium was changed to the indicated conditions and the cells were further incubated for 24 h. Then, O 2 consumption was measured. Oligomycin is an ATP synthase inhibitor; FCCP (carbonilcyanide p -triflouromethoxyphenylhydrazone) is an uncoupler; rotenone is a complex I inhibitor; and antimycin A is a complex III inhibitor. The data represent means ± S.D. ( n = 2). Representative data from two independent experiments are shown. C. Scheme of hydrolysis of FAD and FMN by CD73 and alkaline phosphatase for its uptake into cells, vitamin B 6 metabolism and mitochondria function

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Incubation