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