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: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 is CD73-specific. ( A ) 22E6 binds to HEK293 cells stably transfected with a CD73 expression plasmid (293/CD73) but not to parental HEK293 cells (293). ( B ) 22E6 specifically precipitates CD73 from lysates. ( C ) 22E6 binds to various permanent cancer cell lines and cells isolated from primary ascites. Plain histograms in ( A , C ) = isotype control antibody.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Control

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 inhibits CD73 enzyme activity. ( A ) U138 MG glioblastoma cells were incubated with APCP, 22E6 or an isotype control antibody in the presence of AMP. The amount of inorganic phosphate was measured in a Malachite Green assay. ( B ) The IC50 of 22E6 was calculated to be 0.5 µg/mL, corresponding to approximately 3.5 nM. ( C ) The generation of inorganic phosphate from ADP is CD73-dependent and effectively blocked by 22E6 as shown on MDA-MB231 and GBM20 cells. CD73-negative T47-D do not produce detectable amounts of phosphate. Experiments were performed three times. ** p < 0.01; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Incubation, Control, Malachite Green Assay

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 blocks CD73 enzyme activity on tumor-derived extracellular vesicles. ( A ) TEVs from GBM20 cells were floated into a Optiprep gradient and separated into eight fractions, which were subsequently tested for the presence of the EV markers CD63, CD81 and TSG-101, as well as of CD73. ( B ) Particle numbers in fractions 1 to 8 were counted by NTA, and the protein content was measured in a Bradford assay. ( C ) Fraction 3 contains vesicles which stain positive for CD63, CD81 and CD73, while it is negative for Calnexin indicative for a cell-free preparation. ( D ) APCP and 22E6 block CD73 activity on TEVs from GBM20 cells and ( E ) isolated from primary ascites from a patient with ovarian cancer. ( F ) enzyme activity can be transferred by CD73-positive TEVs from GBM20 cells onto CD73-negative T47-D cells. Experiments were performed at least three times. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Derivative Assay, Bradford Assay, Staining, Blocking Assay, Isolation

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 is a non-competitive inhibitor of CD73. ( A ) Incubation of U138 MG cells with 22E6 and increasing concentrations of AMP resulted in a decreases Vmax. ( B ) The 22E6 antibody but not the 22E6 Fab fragments inhibits CD73 activity. One representative experiment of three is shown. *** p < 0.001; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Incubation, Activity Assay

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 does not inhibit enzyme activity of soluble CD73. ( A ) specific precipitation of recombinant CD73 by 22E6. ( B ) 22E6 does not detectably inhibit enzyme activity of soluble CD73. Error bars = SD; p < 0.001 for APCP, n.s. for Isotype and 22E6. This experiment was performed at least four times.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Recombinant

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 induces downregulation of CD73 on PDX ALL cells in vivo. ( A ) PDX ALL cells express surface CD73 before transplantation. Black line = 22E6; tinted histogram = isotype control. ( B ) CD73 on PDX ALL cells is enzymatically active, and this activity can be inhibited with 22E6 and APCP. ( C ) NSG mice were transplanted with ALL-272 or ALL-1124 cells. Tumor burden was monitored by bioluminescence imaging (BLI). Mice were treated with 100 µg 22E9 twice per week ( n = 5 for ALL-272, n = 6 for ALL-1124; black triangles) or left untreated ( n = 3 for ALL-272, n = 4 for ALL-1124; grey dots). Tumor burden (BLI signal) relative to treatment start was calculated. Data of two independent experiments were integrated. Shown are individual mice (dots) as well as mean ± standard deviation (line). Tumor burdens of ALL-1124 between control and 22E6 treated mice differed significantly at days 35 ( p < 0.05) and 42 ( p < 0.01). At high tumor burden, mice were sacrificed, and cells were isolated. ( D ) Flow Cytometric analysis of isolated xenograft ALL cells from 22E6 treated (solid line) and control mice (dashed line) checking for expression of CD73. Black line = 22E6 anti-CD73; Tinted = control. ( E ) AMPase assay with ALL cell lines testing for enzymatic activity of CD73 of isolated xenograft ALL cells from 22E6 treated and control mice. *** p < 0.001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: In Vivo, Transplantation Assay, Control, Activity Assay, Imaging, Standard Deviation, Isolation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Purinergic ecto-enzyme CD73 is a context-dependent tumor suppressor in colorectal cancer

doi: 10.1016/j.jbc.2025.110864

Figure Lengend Snippet: Bioinformatic analysis on CD73 expression in CRC patients reveal contradicting role of CD73 in colorectal cancer. 203939_at probeset is used for NT5E expression in the GSE39582 dataset. A and B , high NT5E expression is associated with poor overall survival according to Log-rank multiple cut-off # (LRMC) graph ( A ) and Kaplan-Meier (KM) plot ( B ). The expression threshold at which the lowest log-rank p value is obtained (6.786532) within interquartile range was utilized to generate high (n = 372) and low (n = 147) expression groups. C and D , high NT5E expression is associated with poor and recurrence-free survival (RFS) according to according to LRMC graph ( C ) and KM plot ( D ). The expression threshold at which the lowest log-rank p value is obtained (6.961637) within interquartile range was utilized to generate high (n = 342) and low (n = 177) expression groups. E , NT5E expression (average of 11719174_a_at, 11744681_a_at, 11755207_a_at probesets) in healthy individual versus paired normal and tumor samples of colorectal cancer (CRC) patients in GSE44076 microarray dataset. Healthy (n = 50), Control (n = 98) and Tumor (n = 98) (∗∗∗∗ p < 0.0001). F , NT5E expression in primary tumor and normal colon samples in TCGA COAD patient samples. Tumor (n = 456), Control (n = 41) (∗∗∗∗ p < 0.0001) ( G and H ) NT5E expression in paired normal versus tumor samples of TCGA COAD patients in paired dot plot with different presentation formats. I , NT5E expression in patient samples according to the disease stage from TCGA-COAD dataset; Tumor (n = 456), Control (n = 41). J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas. Patient IDs: healthy (3266), CRC-1 (2001), and CRC-2 (2096). Red dashed lines and red arrows indicate epithelial cells ( E ) while white asterixis show stromal (S) areas with high CD73 expression. K , CD73 protein expression in Clinical Proteomic Tumor Analysis Consortium patient samples as measured by mass spectrometry analysis ( p = 3.72654150065602E-11); normal (n = 100), primary tumor (n = 97). Proteomics results presented here obtained from UALCAN platform. # LRMC graph shows gene expression-based cut-offs on the x axis and log-rank p values at each specific cut-off on the y axis. The threshold within the interquartile range that gives the lowest log-rank p value (6.96) was used to categorize low and high expression groups. For all LRMC graphs: blue and red colors indicate association with good and poor prognosis, respectively; vertical dashed lines represent 25th percentile, median and 75th percentile values. Horizontal dashed line indicates 0.05 p value. IHC, immunohistochemistry; RFS, recurrence-free survival; LRMC, log-rank multiple cut-off; KM, Kaplan-Meier; CRC, colorectal cancer.

Article Snippet: J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas.

Techniques: Expressing, Microarray, Control, Immunohistochemistry, Mass Spectrometry, Gene Expression

Journal: The Journal of Biological Chemistry

Article Title: Purinergic ecto-enzyme CD73 is a context-dependent tumor suppressor in colorectal cancer

doi: 10.1016/j.jbc.2025.110864

Figure Lengend Snippet: CD73 is abundantly expressed in cancer cells in tumors and in most CRC cell lines. A and C , expression of CD73 in FACS sorted cells obtained from 6 CRC tumors (donors A – K ). Results from 3 different probesets of CD73 (203939_PM_at, 1553994_PM_at and 1553995_PM_at). ∗Log2 intensities are obtained from normalized microarray data; therefore, the values are absolute not relative. D and F , UMAP based visualization of GSE178318 dataset which contain 6 primary tumor samples of CRC patients. Clusters were either colored according to ( D ) different cell populations or ( E ) NT5E expression levels ( F ) Average NT5E expression in each cluster in UMAP was also visualized via dot plo ts. G , CD73 protein expression levels from seven different CRC cell lines compared to that of two immortalized immune cell lines such as Namalwa (B lymphocytes) and Jurkat (T lymphocytes) via Western blot. H , NT5E mRNA levels were measured by qPCR in the same cell lines that were checked with Western blotting. (I and J ,) cell surface expression profile of CD73 on indicated CRC cells analyzed by flow cytometry, reflecting ( I ) the intensity of CD73 signal (signal-to-noise ratio) and ( J ) its density in the cell population (% of the cells expressing CD73), which is in line with the Western blot result. CRC, colorectal cancer.

Article Snippet: J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas.

Techniques: Expressing, Microarray, Western Blot, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Purinergic ecto-enzyme CD73 is a context-dependent tumor suppressor in colorectal cancer

doi: 10.1016/j.jbc.2025.110864

Figure Lengend Snippet: CD73 expression loss enhances CRC cell proliferation, clonogenicity and anchorage-independent growth and lead to larger xenografts than the ones of their controls. A and B , cell proliferation assay of control or CD73-deficient ( A ) DLD-1 and ( B ) HT-29 cells. Cells were seeded into 96 well plates, triplicate for each condition, then viable cells were quantified every day thereafter by using Cell Tilter Glo kit which measured ATP amount of metabolically active cells. Relative cell growth of each condition was calculated by dividing their values to the ones of the control cell of the first day. C and D , percentage of cell population in S phase of control or CD73-deficient (C) DLD-1 and (D) HT-29 cells from the flow cytometry analysis were graphed. (∗ p < 0.05, ∗∗ p < 0.01) for both cell lines. E and H , colony formation assay and quantification graphs of control or CD73-deficient ( E – G ) DLD-1 and ( F – H ) HT-29 cells. I and J , anchorage-independent colonies of CD73-deficient CRC ( I ) DLD-1 and ( J ) HT-29 cells that were plated onto polyHema-coated 96 well plates and colony formation was pictured over the indicated times. CD73 depleted cells were showing more numerous and bigger colonies as compared to their control cells for both cell lines. K and L , xenograft tumors of control or CD73-deficient ( K ) DLD-1 (sgNT5E-DLD-1) and ( L ) of HT-29 (sgNT5E-HT-29) cells that were injected subcutaneously to the flanks of athymic nude mice. M and N , tumor weights at the time of sacrifice also showed significant differences for both constructs in DLD-1 ( p < 0.001 and 0.0017, respectively) and only in one construct in HT-29 ( p = 0.0829 and 0.0421, respectively). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 for both cell lines. O , Ki67 and H&E histological stainings in xenograft tumor samples of control and CD73-deficient DLD-1 and HT-29 cells (this scale bar represent100 μm). P , proliferation indices based on Ki67 IHC stainings in xenograft tumor samples of control and CD73-deficient DLD-1 and HT-29 cells, represented as bar graphs. IHC, immunohistochemistry; CRC, colorectal cancer.

Article Snippet: J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas.

Techniques: Expressing, Proliferation Assay, Control, Metabolic Labelling, Flow Cytometry, Colony Assay, Injection, Construct, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Purinergic ecto-enzyme CD73 is a context-dependent tumor suppressor in colorectal cancer

doi: 10.1016/j.jbc.2025.110864

Figure Lengend Snippet: CD73 loss of expression promoted migration and invasion of CRC cell lines, involving the EMT process. A and D , wound healing assay and quantitative measurement of the gap closures of control versus CD73-deficient ( A and B ) DLD-1 after 36 h and ( C and D ) HT-29 after 7 days. CD73-deficient cells moved faster than their control group in both cell lines. E and F , transwell Invasion assay with control versus CD73-deficient ( E ) DLD1 or ( F ) HT-29 cells. Transwells were precoated with a thin layer of Matrigel before seeding the cells in 1% Fetal Bovine Serum media. 20% Fetal Bovine Serum media were added to the outer chamber to create cell attraction force. Transwells of DLD1 cells were stained after 24 h, and of HT-29 cells were stained after 48 h. Pictures were taken by camera ( E-up ) and inverted microscope ( E-down and F ) at representative fields of the wells. The CD73-deficient cells were also more invasive as compared to their control groups. G and H , loss of CD73 expression is associated with enhanced EMT signature in DLD1 ( G ) and HT29 ( H ) cells. I and L , Cell morphology and their EMT profile by Western blots of gefitinib resistant DLD1 ( I – J ) and HT29 ( K – L ) cell lines.

Article Snippet: J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas.

Techniques: Expressing, Migration, Wound Healing Assay, Control, Transwell Invasion Assay, Staining, Inverted Microscopy, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Purinergic ecto-enzyme CD73 is a context-dependent tumor suppressor in colorectal cancer

doi: 10.1016/j.jbc.2025.110864

Figure Lengend Snippet: Overexpression of CD73 in CRC cells displays some reversed phenotypes of CD73 loss of expression. A and C. CD73 transient overexpression induced apoptosis in HT-29 cells as seen in ( A ) Western blot and flow cytometry via ( B ) PI (PE/left axis)-Annexin V (FITC/right axis) staining and ( C ) quantitation of this flow cytometry data. D and J , CD73 stable transfection reduces cell growth in vitro . D and E , decrease in cell proliferation upon CD73 overexpression is measured via flow cytometry with cell cycle analysis (PI-staining). F and G , impaired cell migration upon CD73 overexpression is quantified via wound healing assay. H and J , CD73 overexpressing cells also form smaller xenografts in vivo . K , Ki67 and H&E histological stainings in xenograft tumor samples of control and CD73 overexpressing HT-29 cells (scale bar: 100 μm). L , proliferation indices based on Ki67 IHC stainings in xenograft tumor samples of control and CD73-overexpressing HT-29 cells, represented as bar graphs. M , overexpression of CD73 also sensitizes HT-29 cells to gefitinib. N and O , EMT signature was also reverted in both transient (N) and stable ( O ) expression of CD73. ∗Western blot panels in ( A and N ) were generated from the same lysates for each sample. Therefore, a representative loading for β-actin was used for both panels. IHC, immunohistochemistry; CRC, colorectal cancer.

Article Snippet: J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas.

Techniques: Over Expression, Expressing, Western Blot, Flow Cytometry, Staining, Quantitation Assay, Stable Transfection, In Vitro, Cell Cycle Assay, Migration, Wound Healing Assay, In Vivo, Control, Generated, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Purinergic ecto-enzyme CD73 is a context-dependent tumor suppressor in colorectal cancer

doi: 10.1016/j.jbc.2025.110864

Figure Lengend Snippet: CD73 expression affects cell fate of CRC cells by multiple cell death inducers. A and C , CD73 stably expressing HT29 cells (CD73 OE by lentiviral pBABE-NT5E) or ( D – F ) CD73-deficient HT29 cells (CD73 ko by CRISPR/Cas9 (sgNT5E)) were treated with GEF (25 μM, 16 h), Doxorubicin (10 μM, 16 h) and TNF alpha (200 ng/ml, 24 h) respectively. After indicated times for each experiment, cells were harvested and subjected to staining process as described in the section. Each sample has two peaks of fluorescent equivalent to alive and dead cell populations. There are triplicate for each sample in all experiments and the presented results are presentative of at least two independent repeats. Fluorescent graphs for each experiment are just representative of one sample replicate. G and H , representative Western blots showing CD73 expression of ( G ) CD73 stably expressing HT29 cells (CD73 OE ) and ( H ) CD73 depleted HT29 cells (CD73 ko ). ( I ) TUNEL staining of xenograft tumor samples from control or CD73 overexpressing HT-29 cells. J , quantification of TUNEL-positive versus DAPI-only cell percentages in xenografts shown in panel I, represented as bar graph. K , H&E staining (magnified from K ) showing increased number of dead cells in CD73 overexpressing HT-29 (CD73 OE ) xenografts, possibly not undergoing apoptosis. L , TUNEL staining of xenograft tumor samples from control or CD73-deficient (CD73 ko ) HT-29 cells. M , quantification of TUNEL-positive versus DAPI-only cells in xenografts shown in panel L, represented as a bar graph. This scale bar represent 100 μm for all panels. CRC, colorectal cancer.

Article Snippet: J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas.

Techniques: Expressing, Stable Transfection, CRISPR, Staining, Western Blot, TUNEL Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Purinergic ecto-enzyme CD73 is a context-dependent tumor suppressor in colorectal cancer

doi: 10.1016/j.jbc.2025.110864

Figure Lengend Snippet: CD73 impacts CRC patient survival and EMT in tumors depending on immune cell infiltrates and stroma involvement. A and C , High NT5E expression is associated with poor recurrence-free survival in samples only with high immune score (Immune-high) ( C ) yet no such association is observed with immune-intermediate and immune-low groups according to LRMC graphs # ( A and B ). D and I , KM-plots of patients (RFS) according to NT5E expression status (high versus low) in immune-low, -intermediate and -high groups in D and F , GSE39582 and ( G – I ) GSE17536 datasets. High expression of NT5E is significantly associated with shorter RFS only in the immune-high groups ( I ). 203939_at probeset is used for NT5E expression. Median expression is used as a cut-off separately for immune low, int and high groups. J , multivariate cox regression analyses with immune score ## for RFS. K and M , NT5E promotor methylation is correlated with lower NT5E expression and worse prognosis in TCGA COAD patients. K , methylation β levels of the selected CpGs that are located on the transcription start site of the NT5E gene in primary tumor (n = 301) and normal (n = 38) colon samples, (∗ p < 0.05, ∗∗ p < 0.01, ns: not significant). L , scatter plot showing NT5E expression and methylation β levels for cg13315970 CpG. Pearson correlation r and p values are given. M , KM-plot of patients with higher methylation of NT5E promotor at cg13315970 had relatively worse survival, (∗ p < 0.05). # LRMC graph shows gene expression-based cut-offs on the x axis and log-rank p values at each specific cut-off on the y axis. The threshold within the interquartile range that gives the lowest log-rank p value (6.96) was used to categorize low and high expression groups. For all LRMC graphs: blue and red colors indicate association with good and poor prognosis, respectively; vertical dashed lines represent 25th percentile, median and 75th percentile values. Horizontal dashed line indicates 0.05 p value. ## Samples were sorted based on Estimate's immune score. 188, 189 and 189 samples were categorized as immune score low (assigned to 1), intermediate (assigned to 2) and high (assigned to 3), respectively. Assigned values for these categories (1-2-3) were used as continuous variables in multivariate analyses. Patients with available non-zero survival time were included in the analysis. (CI: confidence interval; HR: hazard ratio). CRC, colorectal cancer; KM, Kaplan-Meier; LRMC, log-rank multiple cut-off; RFS, recurrence-free survival.

Article Snippet: J , CD73 protein expression was measured via immunohistochemistry (IHC) using antibodies against CD73 (HPA017357) in normal versus tumor (CRC) samples from The Human Protein Atlas.

Techniques: Expressing, Methylation, Gene Expression