Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Expression and purification of CD73 protein. ( A ) E. coli Origami cells were transformed with pET28a-CD73 plasmid and plated on LB agar containing kanamycin. Six colonies were randomly picked and PCR performed with primers for CD73. Size of the product is 536 bp. L represents 100 bp plus ladder. ( B ) Transformation was further confirmed in two of the positive colonies through restriction enzyme digestion with EcoRI and XhoI, either singly (SD) or together (DD). Size of the insert (i) is 1731 bp and of vector is 5369 bp. U, undigested plasmid; M, 1 kb ladder. ( C ) SDS-PAGE analysis of the expression of rCD73 was performed from cells treated with different concentrations of IPTG and/or temperature and cultured in a shaker incubator at 300 rpm displayed nearly same level of expression in each case within the pellet. M, Molecular weight marker; S, lysate supernatant; P, lysate pellet. ( D ) The expressed protein was mostly found in the inclusion body (IB) which was washed several times to get a pure IB. ( E ) The protein in the inclusion body was solubilized in 8 M urea. ( F ) Representative Coomassie-stained SDS-PAGE gel shows the various fractions followed by the protein present in the elute in lane E. ( G ) rCD73 was purified using a Ni-NTA column in the AKTA Pure System which showed a single band. ( H ) The denatured rCD73 was refolded in a refolding buffer, concentrated using Amicon Ultracentrifuge filter with a 30-kDa cut-off, and then dialyzed to remove urea. Representative Coomassie-stained SDS-PAGE gel depicts a strong band around 63 kDa. FT, flow through; DF, dialyzed fraction. ( I ) The identity of this band was further confirmed using Western blot tested with antibody against anti-CD73. Lysate from mouse brain was used as a positive control, PC. RP, recombinant protein. ( J ) Absorbance spectra of the refolded protein is depicted in comparison to the denatured protein present in the inclusion body. A shift in the absorbance at 365 nm wavelength (see arrow) indicates protein folding. ( K ) The specific activity of the refolded recombinant protein was tested through phosphatase assay. *** p < 0.001, n = 3.

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Expressing, Purification, Transformation Assay, Plasmid Preparation, SDS Page, Cell Culture, Molecular Weight, Marker, Staining, Western Blot, Positive Control, Recombinant, Comparison, Activity Assay, Phosphatase Assay

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Exogenous addition of recombinant CD73 reduces the effect of ATP on LPS-mediated COX-2 expression. ( A , B ) RAW264.7 macrophage cells were treated with ATP (100 µM), ATPγS (100 µM) or NECA (100 µM), alone or in the presence of LPS (10 ng/ml). Cells were harvested after 24 h and COX-2 expression was checked. β-actin was used as a housekeeping control. ( C ) RAW264.7 cells were treated with different doses of LPS (0.01–10 µg/ml) for 24 h. Culture media was then collected to estimate amount of ATP released. * p < 0.05 with respect to untreated cells ( n = 4). ( D , E ) J774A.1 macrophage cells were treated with LPS (10 ng/ml), alone or together with ATP (100 µM), AMP (100 µM) or NECA (100 µM). 24 h later cells were harvested and checked for COX-2 expression. Representative Western blot image is depicted ( D ) and the relative fold change, calculated by normalization of COX-2 expression with that of β-actin, from n = 3 experiments, is depicted in ( E ). * p < 0.05 with respect to control. $ p < 0.05 with respect to LPS-treated cells. ( F , G ) J774A.1 cells treated with LPS, AMP and NECA, alone or in combination were harvested after 4 h for the isolation of mRNA followed by cDNA synthesis. Real time PCR was performed to check the expression of various M1 ( F ) and M2 ( G ) markers ( n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 with respect to untreated cells. ( H ) mRNA was isolated from untreated J774A.1 cells and mouse brain tissue and cDNA synthesis was performed. Real time PCR was performed to check the expression of CD73 and CD39 using specific primers. δC t calculated by normalizing with β-actin ( n = 3). ( I , J ) J774A.1 cells were exogenously treated with rCD73 (300 ng) in the presence of ATP alone or ATP together with LPS. 24 h later cell lysates were collected for checking COX-2 expression. Representative blot shown in (I) while relative fold change calculated from normalized COX-2 expression from at least 7 experiments is given in ( J ). ** p < 0.01, *** p < 0.001 ( n = 7). ( K , L ) Cells were treated with LPS, AMP, or AMP-CP (1 µM), in the presence or absence of exogenously added rCD73 protein. After 24 h, cell lysates were used for checking the expression of COX-2. Representative blot shown in ( K ) with relative fold change from normalized COX-2 ( n = 6) given in ( L ). * p < 0.05, ** p < 0.01, *** p < 0.001 relative to the columns shown in the figure. $ p < 0.05 relative to the same stimulation but without rCD73.

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Recombinant, Expressing, Control, Western Blot, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Overexpression of CD73 in J774A.1 cells reduces COX-2 expression. ( A ) Presence of the insert (CD73 gene) in the pcDNA3.1 vector was confirmed through PCR ( A ), and restriction analysis using EcoRI and XhoI digestion ( B ). L, 1 kb plus ladder; U, undigested plasmid; SD, EcoRI-treated plasmid; DD, double digestion. Size of the insert, 1731 bp; size of vector, 5446 bp. ( C ) Following transfection, expression of CD73 checked by real time PCR. δC t calculated by normalizing with β-actin. ** p < 0.01 with respect to non-transfected cells ( n = 3). ( D , E ) CD73 expression in transfected cells was analyzed through Western blot, and relative densitometry shown after normalization with β-actin ( n = 4). ( F ) Activity of CD73 in cell lysates was checked by measuring the amount of phosphate released using AMP as a substrate. *** p < 0.001 ( n = 4). ( G ) CD73 activity was also measured in transfected cells ( n = 4). J774A.1 cells were transfected with CD73 for 28 h followed by replacing the media with a phosphate-free DMEM. AMP (100 µM), with or without AMP-CP (1 µM), was then added to the media which was collected after 1 h for the analysis of free phosphate. ( H , I ) J774A.1 cells were transfected with an empty vector (pcDNA3.1) or with CD73 for 28 h followed by stimulation with LPS (10 ng/ml), AMP (100 µM), or AMP-CP (1 µM), as indicated, for 24 h, for analysis of COX-2 protein using Western blot ( H ). Densitometric analysis ( n = 6) represents relative change in COX-2 expression as fold change with respect to empty vector-transfected control cells ( I ). * p < 0.05, ** p < 0.01, *** p < 0.001 with respected to indicated conditions. $$ p < 0.01 with respect to empty vector-transfected cells also treated with LPS and AMP. (J, K) CD73-transfected cells were stimulated with both LPS (10 ng/ml) and ATP (100 µM) for 24 h followed by immunocytochemistry using anti-COX-2 antibody (green). Cells were counterstained with the nuclear dye, Hoechst 33,342 (blue). Scale bar, 20 μm. Integrated density of COX-2 positive signal was normalized with that of Hoechst 33,342 signal and represented as % in (K). *** p < 0.001 ( n = 2).

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Over Expression, Expressing, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Control, Immunocytochemistry

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: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Expression and purification of CD73 protein. ( A ) E. coli Origami cells were transformed with pET28a-CD73 plasmid and plated on LB agar containing kanamycin. Six colonies were randomly picked and PCR performed with primers for CD73. Size of the product is 536 bp. L represents 100 bp plus ladder. ( B ) Transformation was further confirmed in two of the positive colonies through restriction enzyme digestion with EcoRI and XhoI, either singly (SD) or together (DD). Size of the insert (i) is 1731 bp and of vector is 5369 bp. U, undigested plasmid; M, 1 kb ladder. ( C ) SDS-PAGE analysis of the expression of rCD73 was performed from cells treated with different concentrations of IPTG and/or temperature and cultured in a shaker incubator at 300 rpm displayed nearly same level of expression in each case within the pellet. M, Molecular weight marker; S, lysate supernatant; P, lysate pellet. ( D ) The expressed protein was mostly found in the inclusion body (IB) which was washed several times to get a pure IB. ( E ) The protein in the inclusion body was solubilized in 8 M urea. ( F ) Representative Coomassie-stained SDS-PAGE gel shows the various fractions followed by the protein present in the elute in lane E. ( G ) rCD73 was purified using a Ni-NTA column in the AKTA Pure System which showed a single band. ( H ) The denatured rCD73 was refolded in a refolding buffer, concentrated using Amicon Ultracentrifuge filter with a 30-kDa cut-off, and then dialyzed to remove urea. Representative Coomassie-stained SDS-PAGE gel depicts a strong band around 63 kDa. FT, flow through; DF, dialyzed fraction. ( I ) The identity of this band was further confirmed using Western blot tested with antibody against anti-CD73. Lysate from mouse brain was used as a positive control, PC. RP, recombinant protein. ( J ) Absorbance spectra of the refolded protein is depicted in comparison to the denatured protein present in the inclusion body. A shift in the absorbance at 365 nm wavelength (see arrow) indicates protein folding. ( K ) The specific activity of the refolded recombinant protein was tested through phosphatase assay. *** p < 0.001, n = 3.

Article Snippet: The primary antibodies used in this study were rabbit anti-COX-2 (Cayman Biochemicals, Ann Arbor, MI), rabbit anti-CD73 (SinoBiologicals), rabbit anti-IκB-α (Santa Cruz Biotechnology, Dallas, TX), rabbit anti-phospho-p42/44 MAPK (detecting endogenous levels of p42/44 only when dually phosphorylated at Thr202 and Tyr204 of Erk1 and Thr185 and Tyr187 of Erk2), and rabbit anti-p42/44 MAPK (both from Cell Signaling Tech, Danvers, USA).

Techniques: Expressing, Purification, Transformation Assay, Plasmid Preparation, SDS Page, Cell Culture, Molecular Weight, Marker, Staining, Western Blot, Positive Control, Recombinant, Comparison, Activity Assay, Phosphatase Assay