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: 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

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Flow cytometry results of CD73-positive ADSCs. B Flow cytometry results of CD73-negative ADSCs. C In vitro, immunofluorescence staining of ADSCs in each group. Red indicates CD73, green indicates VEGF, blue indicates DAPI, scale bar = 20 µm. D Quantitative analysis of immunofluorescence staining results among different groups ( n = 3). E Western blot results of CD73 and VEGF proteins after transfection or APCP treatment in each group. F Quantitative analysis of Western blot results. G CCK-8 assay results among different groups of ADSCs in conditioned medium ( n = 3). H Colony formation assay results among RBSMC in conditioned medium ( n = 3). I Cell migration assay results and quantitative analysis among different groups of ADSCs in conditioned medium ( n = 3). J Wound healing assay results among RBSMC in conditioned medium ( n = 3). Data presented as ±SD. * and # indicates P < 0.05, ** and ## indicates P < 0.01, and *** and ### indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Flow Cytometry, In Vitro, Immunofluorescence, Staining, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Cell Migration Assay, Wound Healing Assay

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Western blot results of CD73 and VEGF in the bladder tissues of NB + CD73⁺ / ⁺ group on days 0, 7, 14, 21, and 28 after treatment. B Quantitative analysis of the Western blot results for CD73 and VEGF ( n = 3). C The results of the mean pressure of the voiding contractions and mean intermicturition interval in each group of rats ( n = 5). D Representative images of cystometrography results for each group of rats ( n = 5). E Masson staining results of bladder tissues in each group of rats. scale bar = 50 µm. F Immunofluorescence staining results for CD73 and VEGF in the bladder tissues of each group. Red indicates CD73, green indicates VEGF, and blue indicates DAPI. scale bar = 20 µm. G Quantitative analysis of smooth muscle content in the bladder (yellow square) of each group ( n = 3). H Quantitative analysis of immunofluorescence staining for CD73 and VEGF ( n = 3). Data presented as ±SD. * and # indicates P < 0.05, ** and ## indicates P < 0.01, and *** and ### indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Western Blot, Staining, Immunofluorescence

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Immunofluorescence staining results of ADSCs in the bladder tissues of each group of rats. Red represents ADSCs, green represents βIII-tubulin, and blue represents DAPI. Scale bar = 20 µm. B Immunohistochemical results of CXCR4 in the bladder tissues of each group. The green arrows indicate regions of high CXCR4 expression. Scale bar = 50 µm. C Immunofluorescence staining results of CD73 and SDF-1 in the bladder tissues of each group. Red represents SDF-1, green represents CD73, and blue represents DAPI. Scale bar = 20 µm. D Quantitative results of ADSCs in the bladder tissues of each group ( n = 5). E Quantitative results of SDF-1 expression in the bladder tissues of each group ( n = 3). F In vitro, western blot results of SDF-1 in each group under conditioned medium. G Quantitative results of SDF-1 expression of each group ( n = 3). H Cell migration assay results of CD73⁺ ADSCs after SDF-1 gene knockdown. I Quantitative results of cell migration assays for ADSCs in each group ( n = 3). Data presented as ±SD. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. . Full-section IHC are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, In Vitro, Western Blot, Cell Migration Assay, Knockdown, Migration

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: CD73 activation upregulates VEGF expression, further stimulating the PI3K/AKT/mTOR pathway to enhance cell proliferation. Simultaneously, it inhibits NFκB phosphorylation, suppressing the NFκB/NLRP3/caspase-1 axis, thereby preventing apoptosis and reducing IL-1β and IL-6 levels. Moreover, activated CD73 increases SDF-1 expression, which interacts with its receptor CXCR4 to direct cell migration to damaged bladder tissue.

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Migration