anti cd47 neutralizing antibody  (Bio X Cell)

Journal: Experimental and Therapeutic Medicine

Article Title: CD47 antibody protects mice from doxorubicin-induced myocardial damage by suppressing cardiomyocyte apoptosis

doi: 10.3892/etm.2022.11277

Figure Lengend Snippet: aCD47 significantly reduces the severity of Dox-induced dilated cardiomyopathy (DCM) in mice. Adult mice (aCD47/Dox group) were i.p. administered with both 7 mg/kg aCD47 and 10 mg/kg Dox once a week for 4 weeks. Mice treated with both IgG isotype and Dox (IgG/Dox group) or PBS and aCD47 alone (PBS and aCD47 groups) were controls. (A) Hematoxylin and eosin (H&E) staining for mouse heart tissues. Representative gross morphology and phase contrast microscope images with x200 magnification (upper two panels). Masson staining for interstitial fibrosis and immunostaining for collagen I (lower two panels). Representative images with x200 magnification. Red arrow, cells with centrally localized nuclei; blue arrow, broken and patchy myofibers; yellow arrow, interstitial fibrosis; green, collagen I. (B) Representative echocardiograms of mice 4 weeks after treatment. (C) Quantitative analysis of echocardiographic measurements. Left ventricular ejection fraction (LVEF); left ventricular fractional shortening (LVFS). (D) Quantitative analysis of cardiomyocyte area after H&E staining (upper panel; Mann-Whitney test) and fluorescence intensity of collagen I-positive fibers in heart tissues after immunostaining by ImageJ software. Data are presented as relative fluorescence intensity of positively stained cells over untreated controls. (E) Western blot analysis for the expression of collagen I, Bax and Bcl-2 in the cardiac tissues of treated mice. GAPDH was internal loading control. One representative blot. (F) Band densitometric intensity was semi-quantified by ImageJ software. (G) Flow cytometry analysis for the infiltrating CD11b + macrophages in cardiac tissues. (H) The infiltrating CD11b + macrophages in cardiac tissues were measured by flow cytometry and quantitatively analyzed. All quantitative data are presented as mean ± standard error. * P<0.05, ** P<0.01 vs. PBS group; # P<0.05, ## P<0.01 vs. IgG/Dox group (n=5-7). Two-way ANOVA with Tukey's multiple comparison's test, except where it is indicated otherwise. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin.

Article Snippet: Anti-CD47 neutralizing antibody (aCD47) was purchased from Bio X Cell.

Techniques: Staining, Microscopy, Immunostaining, MANN-WHITNEY, Fluorescence, Software, Western Blot, Expressing, Control, Flow Cytometry, Comparison

Journal: Experimental and Therapeutic Medicine

Article Title: CD47 antibody protects mice from doxorubicin-induced myocardial damage by suppressing cardiomyocyte apoptosis

doi: 10.3892/etm.2022.11277

Figure Lengend Snippet: aCD47 suppresses the expression of cardiac myofiber early apoptosis and production of pro-inflammatory cytokines in mice with Dox-induced DCM. (A) Cardiac myofiber apoptosis in the treated mice was analyzed by flow cytometry analysis. Annexin V + 7-AAD - cells were identified as early apoptotic cells. Annexin V + 7-AAD + cells were identified as late apoptotic cells. Representative dot plot. (B) Quantitative analysis of apoptotic cells after flow cytometry analysis. ** P<0.01 vs. untreated mice; ## P<0.05 vs. mice treated with Dox alone, n=5-7. Two-way ANOVA with Tukey's multiple comparison's test. (C) Different doses of aCD47 (3.5, 7 and 14 mg/kg) on apoptosis of cardiac myofiber in mice with DCM. Representative dot plot (left panel). Quantitative analysis of early apoptotic cells (right panel). * P<0.05 and ** P<0.01 vs. untreated mice; # P<0.05 vs. mice treated with Dox alone (Dox/0 group), n=3/group. Two-way ANOVA with Tukey's multiple comparison's test. (D) Measurement of LDH release in serum. Data are presented as mean ± standard error (Mann-Whitney test). (E) Quantitative analysis of Bax and Bcl-2 expression in cardiac tissues of treated mice in . Data are presented as the ratio of band densitometric intensity over untreated control. (F) Ratio of Bcl-2/Bax expression in lung tissues of treated mice analyzed by western blot analysis in . (G) Immunostaining for Bax (red) in the cardiac tissues of treated mice. Representative images with x200 magnification. (H) Quantitative analysis for Bax expression in the stained cells by ImageJ software. Data are presented as the fluorescence intensity over controls. (I and J) ELISA analysis for the expression of TNF-a in serum and IL-6 in heart protein extracts. Data are presented as mean ± standard error. ** P<0.01 vs. PBS group; # P<0.05, ## P<0.01 vs. IgG/Dox group (n=5-7). Two-way ANOVA with Tukey's multiple comparison's test, except where it is indicated otherwise. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin; LDH, lactate dehydrogenase.

Article Snippet: Anti-CD47 neutralizing antibody (aCD47) was purchased from Bio X Cell.

Techniques: Expressing, Flow Cytometry, Comparison, MANN-WHITNEY, Control, Western Blot, Immunostaining, Staining, Software, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: CD47 antibody protects mice from doxorubicin-induced myocardial damage by suppressing cardiomyocyte apoptosis

doi: 10.3892/etm.2022.11277

Figure Lengend Snippet: aCD47 reduces Dox-induced cardiomyocyte early apoptosis. The cultured primary cardiomyocytes were pre-treated with 1 µg/ml aCD47 for 1 h, which was followed by treatment with 10 µM Dox (aCD47/Dox group) for 24 h. The cells treated with both Dox and isotype IgG (IgG/Dox), untreated or treated with aCD47 alone (aCD47 group) were controls. (A) Flow cytometry analysis for Annexin V + 7-AAD - apoptotic cardiomyocytes 24 h after treatment. Representative dot plots. (B) Quantitative analysis for apoptotic cardiomyocytes analyzed by flow cytometry. (C) Cell viability was determined by CCK-8 assay. Data are presented as the ratio of optical density at 450 nm (OD450 nm) over untreated control. (D) LDH release into the supernatant of the treated cells. Data are presented as the fold increase over untreated controls. Bar plot data in all panels represent the mean ± standard error. n=3. ** P<0.01 vs. 0 group; # P<0.05 vs. IgG/Dox group. Two-way ANOVA followed by a Tukey's multiple comparison's test. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin; LDH, lactate dehydrogenase.

Article Snippet: Anti-CD47 neutralizing antibody (aCD47) was purchased from Bio X Cell.

Techniques: Cell Culture, Flow Cytometry, CCK-8 Assay, Control, Comparison

Journal: Experimental and Therapeutic Medicine

Article Title: CD47 antibody protects mice from doxorubicin-induced myocardial damage by suppressing cardiomyocyte apoptosis

doi: 10.3892/etm.2022.11277

Figure Lengend Snippet: aCD47 reduces the expression of Bax and p-p38 MAPK in Dox-treated cardiomyocytes. (A) Immunostaining for the expression of cTnT, cleaved caspase-1/3, Bax and p-p38 MAPK in the treated cardiomyocytes. Cardiomyocytes were identified as cTnT-positive cells (green). Red, cells positively stained for cleaved caspase-1/3, Bax and p-p38 MAPK. Representative images with x200 magnification. (B) Semi-quantitative analysis of positively stained cells by ImageJ software. Data are presented as the relative intensity of positively stained cells over untreated controls. (C) Western blot analysis for the expression of pro-caspase-1, cleaved caspase-1, cleaved caspase-3, Bax and Bcl-2 in the treated cardiomyocytes. GAPDH was internal loading control. One representative blot. (D) p-p38 MAPK + cells after Dox and aCD47 treatment were analyzed by flow cytometry. Data are presented as the percentage of p-p38 MAPK + cells. (E) Association between the percentage of p-p38 MAPK + cells and apoptotic cardiomyocytes after treatment. Bar plot data in all panels are represented as mean ± standard error. n=3. * P<0.05, ** P<0.01 vs. 0 group; # P<0.05, ## P<0.01 vs. IgG/Dox group. Two-way ANOVA followed by a Tukey's multiple comparison's test. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin; p-, phosphorylated; cTnT, cardiac troponin T.

Article Snippet: Anti-CD47 neutralizing antibody (aCD47) was purchased from Bio X Cell.

Techniques: Expressing, Immunostaining, Staining, Software, Western Blot, Control, Flow Cytometry, Comparison

Journal: Science Advances

Article Title: Zeb1 induces immune checkpoints to form an immunosuppressive envelope around invading cancer cells

doi: 10.1126/sciadv.abd7455

Figure Lengend Snippet: ( A ) H&E at P190 showing clusters of invading cells (arrow). ( B to B″ ) Zeb1 and PDL1 on an invading cluster. Arrows show the same position. ( C ) CD8 cells are excluded from invading clusters (arrow). ( D to D″ ) CD47 and Zeb1 on invading clusters. White arrows show the same position. ( E ) iNos + cells are excluded from invading clusters. ( F to F″ ) Arg1 + cells are closely apposed to Zeb1 hi cells in invading clusters. ( G ) CD8 cells and iNos+ cells surrounding tumors. ( H ) Microscopic views (~200 μm in diameter) were counted for three invasive clusters in three tumors (region 2). Regions at the edge of the tumor 300 μm on either side of an invading cluster were counted and averaged (region 1), as were regions in the central tumor (C). Error bars are SDs. Scale bars, 100 μm. ( I ) Real-time PCR. Primary cells cultured from K-Ras–driven mouse tumors (393P), and cells overexpressing Zeb1 and where Zeb1 was knocked down ( , , ). ( J ) Chromatin immunoprecipitation (ChIP) showing Zeb1 binding to the promoters of CD47 and PDL1 (see fig. S4). “Input” indicates a 1:10 dilution of chromatin before immunoprecipitation. Results are representative of three separate experiments.

Article Snippet: Cells were cocultured in the absence or presence of neutralizing anti-CD47 monoclonal antibody (mAb, 10 μg/ml) or control mAb (BioXCell) for 48 hours and then immunostained as described.

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation

Journal: Science Advances

Article Title: Zeb1 induces immune checkpoints to form an immunosuppressive envelope around invading cancer cells

doi: 10.1126/sciadv.abd7455

Figure Lengend Snippet: ( A to B″ ) Zeb1 + and PDL1 + cells at the IF and invading lung parenchyma (PI) at P220. ( C ) CD8 cells surrounding tumors at P220. ( D and D′ ) CD8 cells at the IF and PI, but not C. ( E ) PDL1 in a Zeb1 mutant background at P220. ( F and G ) CD8 + and PD1 + cells in Zeb1 mutant tumors at P220. ( H to I′ ) CD47 and Zeb1 on cells at the IF and PI. ( J ) Arg1 + cells surrounding tumors at P220. ( K and K′ ) Arg1 + cells adjacent to Zeb1 hi tumor cells (arrows). ( L to L″ ) Close proximity of CD47 + and Arg1 + cells at the IF at P220. ( M ) iNos + cells in normal lung surrounding tumors and airway infiltrate at P220. ( N ) Few iNos + cells are evident at the IF at P220. ( O ) iNos + cells in tumors in a Zeb1 mutant background. ( P to P‴ ) Most CD68 + macrophages in Zeb1 mutant tumors express iNos. ( Q ) Few Arg1 + cells are evident in Zeb1 mutant tumors. ( R ) Effect of Zeb1 on cells in the C, IF, and PI regions of tumors. Regions were identified and counted as in . Error bars are SDs. Scale bars, 100 μm. nd, not detected.

Article Snippet: Cells were cocultured in the absence or presence of neutralizing anti-CD47 monoclonal antibody (mAb, 10 μg/ml) or control mAb (BioXCell) for 48 hours and then immunostained as described.

Techniques: Mutagenesis

Journal: Science Advances

Article Title: Zeb1 induces immune checkpoints to form an immunosuppressive envelope around invading cancer cells

doi: 10.1126/sciadv.abd7455

Figure Lengend Snippet: ( A ) H&E section showing invasion of an AW at P220. ( B to B⁗ ) Coinduction of Zeb1 and CD47 at sites of AW invasion. ( C to E″ ) Close proximity of cells expressing CD47 and Arg1 at sites of AW invasion. ( F ) Close proximity of Zeb1 hi AC and Arg1 + macrophages at sites of AW invasion. ( G ) Quantification of cells expressing Zeb1, CD47, iNos, and Arg1 within AWs and surrounding AWs. Immunostaining in Zeb1 mutant lesions and for iNos is not shown. Three representative microscopic views (~200 μm in diameter) were counted for each region within a single tumor. In addition, results from three tumors from different lungs were averaged. As noted above, Zeb1 mutation prevents AD-to-AC transition, and persisting AD do not show evidence of AW invasion or induction of CD47 around AWs. In addition, note Zeb1 is not expressed in AD cells. Error bars are SDs. Scale bars, (A to D‴ and F) 100 μm and (E to E″) 25 μm.

Article Snippet: Cells were cocultured in the absence or presence of neutralizing anti-CD47 monoclonal antibody (mAb, 10 μg/ml) or control mAb (BioXCell) for 48 hours and then immunostained as described.

Techniques: Expressing, Immunostaining, Mutagenesis

Journal: Science Advances

Article Title: Zeb1 induces immune checkpoints to form an immunosuppressive envelope around invading cancer cells

doi: 10.1126/sciadv.abd7455

Figure Lengend Snippet: ( A ) Bone marrow macrophage (BMM) was cocultured with 393-P cells. Cells were treated with IL-4 to drive M2-like polarization. mRNA levels were quantified by real time PCR. ( B and C ) Cocultures were immunostained for Arg1 or CD206. White arrows show larger 393-P nuclei and yellow arrows show smaller BMM nuclei. ( D and E ) Arg1 and CD206 in BMM treated with IL4. ( F and G ) Cocultures in the presence of CD47 blocking antibody (10 μg/ml). ( H and I″ ) Nomarski images and immunostaining of adjacent macrophages and cancer cells. ( J ) Quantification of results in (A) to (I). Error bars are SDs. ( K ) Real-time PCR showing that IL-4 induces PDL1 mRNA in BMM. Three experiments are shown. ( L to M″ ) Arg1 + and PDL1 + cells at the IF and iNos + and PDL1 − cells in the central region of a mouse lung tumor at P220 (C). ( N ) Quantification of cells in (L) to (M″). Four tumors were analyzed. Scale bars, (H to I″) 10 μm and (other panels) 50 μm. ( O ) Left: A mouse lung tumor is shown with invading cells expressing the mesenchymal marker Col1a2 (green). Right: The image was colorized with red depicting inflammatory and green immunosuppressive areas.

Article Snippet: Cells were cocultured in the absence or presence of neutralizing anti-CD47 monoclonal antibody (mAb, 10 μg/ml) or control mAb (BioXCell) for 48 hours and then immunostained as described.

Techniques: Real-time Polymerase Chain Reaction, Blocking Assay, Immunostaining, Expressing, Marker