Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: The characteristics of ERCs and the adenosinergic enzymatic activity of CD73 on ERCs in vitro . (A) ERCs were detected by flow cytometry to detect negative markers (CD79a and HLA-DR) and positive markers (CD44 and CD105) on cell surfaces for identification. (B) Flow cytometry reflected CD73 expression on the membrane of ERCs and CD73 -/- ERCs. (C) Western blot analysis showed the differences in CD73 protein expression in ERCs and CD73 -/- ERCs. (D) Gray value analysis with immunoblot based; CD73 intensity analysis was homogenized after comparing to β-actin (n=3). (E) Levels of inorganic phosphate (Pi) were measured after adding CD73 substrate (5′ AMP; 1 mM) (ERCs vs . CD73 -/- ERCs: 60.46 ± 4.647 μM vs . 2.097 ± 0.1614 μM, P < 0.0001, n=6). Statistical analysis was done by using unpaired two-tailed Student’s t-tests. Data are presented as mean ± s.e.m (SEM). ****P < 0.0001, analyzed by unpaired t test. ERCs, endometrial regenerative cell; CD73 -/- ERCs, ERCs transfected with lentivirus.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Activity Assay, In Vitro, Flow Cytometry, Expressing, Membrane, Western Blot, Two Tailed Test, Transfection

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: Knockout of CD73 reverses the therapeutic effect of ERCs on colitis. (A) In vivo , BLI of ERCs were tracked at 6, 24, 48, and 72 hours after intraperitoneal or tail intravenous injection in DSS-induced colitis mice. (B) Daily body weight changes in each group for 10 days (n = 6). (C) Representative pictures showed blood stools in BALB/c mice on day 10 after DSS induction. (D, E) Representative photographs of the colon in each group and the colon’s length in each group were analyzed on day 10 (n = 6). (F) Representative H&E-stained micrographs of the colon; blue arrows indicate epithelial detachment, red arrows indicate epithelial hyperplasia, black arrows indicate infiltrating inflammatory cells, and black boxes indicate the loss of enteric crypts (scale bar: 200 μm) (n=6). (G) Histopathology scores were calculated to assess colonic damage quantitatively. Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Knock-Out, In Vivo, Injection, Staining, Histopathology, Comparison

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: Knockout of CD73 reverses the effect of ERCs on intestinal barrier restoration in colitis. (A) Alcian blue staining showing goblet cells (blue) in colon tissue (Scale bars: 400 or 100μm). (B) Quantitative analysis indicates the number of goblet cells in colon tissue (n = 6). (C) Representative TEM images show colonic epithelial cells’ ultrastructure (scale bar:1μm). Red arrows indicate colonic cell microvilli, and blue arrows outline tight junctions and the intercellular space between two adjacent cells. (D) The concentration of serum FITC-dextran suggests the intestinal mucosa’s permeability in each group (n=6). (E-H) Quantitative analysis of intestinal interepithelial tight junction proteins ZO-1, Occludin, and Claudin-1 with β-actin as a reference in colon tissues measured by western blot (n=3). Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Knock-Out, Staining, Concentration Assay, Permeability, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: Knockout of CD73 reverses the effect of ERCs on CD4 + T cell differentiation in colitis. MLN cells were collected on day 10 after DSS induction. To identify Th1 and Th17 cells, MLN cells were first incubated with a stimulation cocktail for 5 h, followed by staining with a fluorescent antibody. Flow cytometry plots and graph analysis of CD4 + IFN-γ + Th1 (A) , CD4 + IL-17 + Th17 (B) , and CD4 + CD25 + Foxp3 + Tregs (C) in the MLNs from the Control, untreated, ERCs-treated and CD73 -/- ERCs-treated groups. (D-F) Percentage of CD4 + IFN-γ + Th1, CD4 + IL-17 + Th17 cells and CD4 + CD25 + Foxp3 + Tregs (n=5). Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Knock-Out, Cell Differentiation, Incubation, Staining, Flow Cytometry, Control, Comparison

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: Knockout of CD73 reverses the effect of ERCs on cytokine profile in colitis. To evaluate the role of CD73 expression on ERCs in regulating immune response during intestinal inflammation at both quality and quantity levels, the production of pro-inflammatory cytokines (IL-1β, IFN-γ and TNF-α) and anti-inflammatory cytokines (IL-10) in colonic tissues was detected via ELISA assay. On day 10, the levels of pro-inflammatory (A-C) and anti-inflammatory (D) cytokines in the colonic tissues were analyzed (n=6). Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Knock-Out, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: Knockout of CD73 reverses the effect of ERCs on preventing DC maturation in colitis. To determine whether each treatment affects DC phenotypes, anti-CD11c antibody and antigen presenting-related antibodies (anti-MHCII, anti-CD86) were used to measure mature DCs in MLNs. (A, B) Representative dot plots of CD11c + CD86 + DCs and CD11c + MHCII + DCs in MLNs. (C, D) Percentage of CD11c + CD86 + DCs and CD11c + MHCII + DCs, respectively (n=6). (E) Infiltration of CD11c + cells was determined by immunohistochemical staining in the colon tissues of each group (Scale bar: 50 μm). (F) Percentage of CD11c + cells by Image J analysis (n = 6). Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Knock-Out, Immunohistochemical staining, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: Knockout of CD73 reverses the effect of ERCs on decreasing the percentage of mature DCs and enhancing the tolerogenic function of the DCs in vitro . ERCs and CD73 -/- ERCs were co-cultured with immature DCs obtained from the bone marrow of BALB/c mice with 200ng/ml LPS for 24 hours in a transwell system. The percentage of CD11c + CD86 + DCs and CD11c + MHCII + DCs was measured by flow cytometry analysis. (A, B) Representative dot plots of CD11c + CD86 + DCs and CD11c + MHCII + DCs. (C, D) Percentage of CD11c + CD86 + DCs and CD11c + MHCII + DCs (n=6). (E-H) The IL-6, TNF-α, IL-1β, and IL-10 were examined in the supernatant of co-cultured cells (n=6). (I) Contour plot of Ki-67 + T cells in vitro. (J) Percentage of Ki-67 + cells (n=4). Data are presented as mean ± s.e.m (SEM). ns, no significance; * P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Knock-Out, In Vitro, Cell Culture, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis

doi: 10.3389/fimmu.2023.1155090

Figure Lengend Snippet: Knockout of CD73 reverses the effect of ERCs on the tolerogenic function of the DCs associated with the STAT-3 pathway in vitro . To explore whether STAT3 would participate in DC maturation and activation mediated by CD73 expressing ERCs in vitro , western blot was used to determine p-STAT3 and STAT3 with β-actin as a reference in DCs (A-C, n=3). Data are presented as mean ± s.e.m (SEM). ns, no significance; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight.

Techniques: Knock-Out, In Vitro, Activation Assay, Expressing, Western Blot, Comparison

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 2. BsAb CD73xEpCAM has dual binding specificity for CD73 and EpCAM. (A) Topology of expression plasmid pbsAb CD73xEpCAM-IgG2s. (B) Schematic depiction of bsAb CD73xEp CAM. (C) bsAb CD73xEpCAM (lane 1 and 2) or oleclumab (lane 3 and 4) (5 μg each) were separated by an SDS-PAGE gel using non-reducing (NR) and reducing (R) conditions. Under non- reducing conditions CD73xEpCAM migrated as a single protein band with an apparent molecular weight of 175 kDa (lane 1), which dropped to 75 kDa when separated under reducing condi tions (lane 2). This is in good agreement with the calculated molecular weight of 83 kDa for CD73xEpCAM monomer and the proposed disulfide-stabilized dimeric single chain composition of the native protein. Oleclumab showed the expected heterodimeric composition of heavy and light chain characteristic for conven tional antibodies (lane 3 and 4). The protein bands of bsAb CD73xEpCAM are indicated by solid black arrows and bands of oleclumab by open black ar rows. (D) Comparison of dose-dependent binding of bsAb CD73xEpCAM, bsAb CD73xMock and bsAb MockxEpCAM to CHO, CHO.EpCAM and CHO.CD73 cells, respectively. (E) Competitive binding assay in which bsAb CD73xEpCAM was pretreated with excess amounts of solu ble CD73 (sCD73), soluble EpCAM (sEpCAM) or a combination thereof (10 μg) prior to incubation with H292 can cer cells. Graphs D-E were analyzed by flow cytometry. All graphs represent mean ± SD. Statistical analysis in graphs E was performed using un-paired T test (****p < .0001). M = marker in graph C.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Binding Assay, Expressing, Plasmid Preparation, SDS Page, Molecular Weight, Comparison, Competitive Binding Assay, Incubation, Flow Cytometry, Marker

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 1. CD73 and EpCAM are highly abundant on EVs. (A) Representative images of immunoblot anal ysis for presence of CD73, EpCAM, PD-L1, CD9, TSG101, calnexin and β-actin in cancer cells H292, OvCAR3, DLD1 and corresponding EVs. (B) CD73, EpCAM and TSG101 in EVs derived from parental H292, H292CD73−KO and H292EpCAM−KO cells, respectively. 20 μg protein of each sample (both cells and EVs) was loaded. Of note: The apparent differ ence in EpCAM signal in H292 cells between Fig. 1A and B is due to a reduction of exposure time during bioluminescent-based detection to prevent over exposure of the very high EpCAM signal from H292- derived EVs in Fig. 1B.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Western Blot, Derivative Assay

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 3. BsAb CD73xEpCAM inhibits the enzyme activity of CD73 on cancer cells and EVs in an EpCAM-directed manner. (A–B) Increasing amounts of H292 and H292CD73−KO cancer cells or EVs were incubated with AMP (100 μM) and inorganic phosphate (Pi) produced by CD73- mediated hydrolysis of AMP was evaluated. (C–D) BsAb CD73xEpCAM, bsAb-controls or oleclumab (1 μg/ml) were added to H292 cancer cells or EVs and evaluated for capacity to inhibit the enzyme activity of CD73. Background levels of Pi in the absence of bsAb CD73xEpCAM was used to normalize CD73 inhibition to 0%. (E) Competitive CD73 enzyme inhibition assay in which bsAb CD73xEpCAM was pretreated with excess amounts of sEpCAM (10 μg) prior to incubation with H292-derived EVs and assessed for its ca pacity to inhibit the enzyme activity of CD73. (F) EpCAM- directed blockade of CD73 on H292-derived parental, EpCAM-KO and CD73-KO EVs using bsAb CD73xEpCAM (1 μg/ml). CD73-mediated hydrolysis of AMP into ADO was evaluated using a colorimetric malachite green-based Pi assay. All graphs represent mean ± SD. Statistical analysis in graphs C-D was performed using un-paired T test. Statistical analysis in graph E was performed using one-way ANOVA followed by a Tukey post-hoc test. Statistical analysis in graph F was performed using multiple T tests (*p < .05, **p < .01, ***p < .001, ****p < .0001).

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Activity Assay, Incubation, Produced, Inhibition, Enzyme Inhibition Assay, Derivative Assay

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 5. BsAb CD73xEpCAM restores the anticancer activity of EV-suppressed PBMCs. (A) PBMCs were cultured in the presence, or absence, of H292EVs or H292 CD73-KOEVs (50 μg/106 PBMCs) at 37 ◦C for 3 d. Next, PBMCs were incubated (or not) with AMP (100 μM) at 37 ◦C for 24 h. Subsequently, cytotoxic T (Effector) cells were stimulated and re-directed to kill EpCAM-expressing PC3M (Target) cancer cells using BIS-1 in an effector (E) to target (T) cell ratio of 4:1. Subsequently, effector and target cells were co- cultured for 2 d in the presence of a conditionally fluorescent caspase 3/8–488 probe. Live cell imaging technology was used to evaluate induction of apoptotic cancer cell death (caspase-3/8 activation, count per image) by taking pictures every 1.5 h at 10x magnification at 37 ◦C for 2 d. (B) PBMCs were incubated with H292EVs (50 μg/106 PBMCs) in the present or absence of bsAb CD73xEpCAM, bsAb controls or oleclumab (1 μg/ml) at 37 ◦C for 3 d. Subsequently, PBMCs were incubated (or not) with AMP (100 μM) at 37 ◦C for 24 h and re-directed using BIS-1 to kill PC3M cancer cells. Using live cell imag ing technology, apoptotic cell death was analyzed over time. (C) IFN-γ levels in culture supernatant of graph B were measured by ELISA. All graphs repre sent mean ± SD. Statistical analysis in graph C was performed using un-paired T test (****p < .0001). Ole = oleclumab in B and C.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Activity Assay, Cell Culture, Incubation, Expressing, Live Cell Imaging, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 6. BsAb CD73xEpCAM inhibits the enzyme activity of CD73 on cancer patient-derived EVs. (A) Characterization of colon carcinoma patient- derived EVs. (B) Representative immunoblotting im ages of the detection of CD73, EpCAM and TSG101 in cancer patient-derived EVs (20 μg). (C) Cancer patient-derived EVs were incubated with AMP (100 μM) and Pi produced by CD73-mediated hydrolysis of AMP was evaluated. (D) BsAb CD73xEpCAM, bsAb- controls or oleclumab (1 μg/ml) were added to EVs derived from patient # 6 and evaluated for capacity to inhibit the enzyme activity of CD73. Background levels of Pi present in the medium in the absence of bsAb CD73xEpCAM was used to normalize CD73 in hibition to 0%. All graphs represent mean ± SD. Statistical analysis in graph D was performed using un-paired T test (*p < .05, ***p < .001).

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Activity Assay, Derivative Assay, Western Blot, Incubation, Produced