Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from . P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from ). P-values were determined by two-sided Student’s t-test.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Infection, Quantitative Proteomics, Control, Virus, Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 aging” (A) and “CD47 hypertension” (B). C) Overview figure of the data derived from the literature searches. Age-related increased CD47 levels may contribute to pathogenic conditions associated with severe COVID-19.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay, Infection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: CD47 is overexpressed on malignant lymphocytes in mycosis fungoides (MF) tumors. a A representative image of a skin involved by MF demonstrates intense CD47 staining on atypical TOX + malignant cells. HE, hematoxylin and eosin (20x). b Targeted single-cell RNA transcriptomics as tSNE plots of concatenated tumors from three patients with MF tumors (n = 287 cells total). Clusters called by recursive dendrogram split and annotated from preferentially expressed genes. Tcm, T cell central memory; Tem, T cell effecor memory; DC, dendritic cells. c The intensity of CD47 expression (anti-CD47 antibody-oligo conjugate; AbSeq) over the various cell population defined in Fig. 1b. d Statistical analysis of expression of CD47 (molecules per cells) in different cell populations. *, p < 0.05; ***, p < 0.001 e CD47 expression on CD3 + TOX + MBL2 cells. Flow cytometry of a cell suspension from a primary cell culture. Grey tinted area, an isotype control. Red tinted area, anti-CD47 antibody. f Tumor growth curves of CD47hi WT MBL2 (WT) and CD47 KO MBL2 (CD47 KO) after implantation in B6.SJL mice. n = 5 mice in each group. g Representative imaging of mice 10 days after implantation of CD47hi WT MBL2 (WT) or CD47 KO MBL2 (KO) cells demonstrating large ulcerated tumor in WT, while KO mouse exhibited medium-size tumor without ulceration

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Staining, Expressing, Flow Cytometry, Suspension, Cell Culture, Control, Imaging

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: Anti-CD47 therapy is efficient in controlling the malignant lymphoma growth in a murine model of mycosis fungoides (MF). a Design of anti-CD47 experiments (I.P., intraperitoneal). b Tumor thickness after MBL2 implantation during treatment with anti-CD47 antibody or irrelevant IgG control (n = 5 mice per group). c Percentage of TOX + tumor cells in the inflammatory infiltrate of auricular skin at day 24 after implantation. The percentage of caspase-3 + cells is indicated in black (n = 5 mice per group) (Tx, treatment). d Percentage of F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). e Percentage of MHC class II + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). f Percentage of TNF-α + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). g Percentage of NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). h Percentage of IFN-γ + cells among NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). i Percentage of CD62L + NKG2A-cells among NK1.1. + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). j Schematic of experimental design. k Representative images of mice with tumors after treatment with IFN-α, anti-CD47, or combination anti-CD47 + IFN-α. l The percentage of malignant cells (TOX +) in the TME on day 24 after implantation of MBL2 (n = 5 mice per group). m Representative flow cytometry of CD107a and IFN-γ NK cells (gated on NK1.1 + cells) during therapy with anti-CD47 antibody. n Quantification of CD107a + IFN-γ + NK cells and CD107a + IFN-γ-NK cells (n = 5 mice per group). o The cytotoxic assay of NK cells derived from splenocytes of treated mice co-cultured with MBL2 cells at the indicated effector to target cell ratios

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Control, Flow Cytometry, Derivative Assay, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The effect of anti-CD47 therapy is mediated by cytotoxic NK cells and does not depend on IFN-γ. a Schematic of NK1.1 depletion experiment. b A representative flow showing the percentage of NK cells in non-depleted and NK-depleted mice prior to therapy. c Representative images of mice with tumors 14 days after MBL2 implantation with and without NK1.1 depletion prior to therapy initiation. D The percentage of malignant cells (CD3 + TOX + cells) per ear in non-depleted and NK-depleted mice 24 days after MBL2 implantation. N = 5 mice per group. ***, p < 0.001 e Tumor thickness 24 days after MBL2 implantation in mice treated with irrelevant IgG or anti-CD47 antibody (n = 5 mice per group). f Schematic of experimental design for IFN-γ KO mice. g The volume of lymph nodes 24 days after MBL2 implantation in mice treated with anti-CD47 antibody (n = 3 mice per group). *,p < 0.05; **,p < 0.01; ****,p < 0.0001; ns, non-significant

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The anti-CD47 therapy is accompanied by an influx of NK cells in the TME in patients with relapsed/refractory mycosis fungoides (MF). a Representative images of high and low CD56 number in the dermal infiltrate of tumor MF. b Inverse correlation of the number of CD56 cells per 100 dermal lymphocytes and histoscore of CD47 on the epidermotropic malignant lymphocytes (n = 17, r = – 0.48, p < 0.05). c Representative images of a patient treated with six intra-tumoral injections of 10 mg TTI-621 demonstrate a significant reduction of all tumors and plaques after treatment (I, injected tumor; C, control non-injected tumor). d Multispectral fluorescent immunohistochemistry of CD3, CD56, TOX, and DAPI staining of representative tumors before and after treatment with TTI-621 (60X). e Percentage of malignant cells (CD3 + CD4 + TOX +) and NK cells (CD3-CD56 +) in the TME (n = 11 patients; 4 responders and 7 non-responders). f tSNE analysis of cellular composition in patients with cutaneous T cell lymphoma who had responded to therapy with TTI-621 (n = 4 patients), showing eight distinct clusters of NK cells. g Phenotypic characterization of eight distinct clusters of NK cells depending on the percentage of markers. h Grouping of eight NK cell clusters in two groups based on high vs. dim CD56 expression. i Changes in CD56high NK cells vs. CD56dim NK cells after intra-tumoral injection of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4). j Percentage of NK cells before and after six intra-tumoral injections of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4).. *,p < 0.05; **,p < 0.01

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Injection, Control, Immunohistochemistry, Staining, Expressing

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Histograms depicting cell surface expression of CD24 and CD47 by flow cytometry on mouse cancer cell lines. B, Correlation of CD24 and CD47 surface expression of cell lines shown in A by geometric MFI. Data shown as mean ± SD of 3 technical replicates. Simple linear regression was performed to assess correlation. C, Representative plots showing quantification of CD45+ phagocytic primary mouse macrophages co-cultured with CFSE+ KPCA.C. Co-cultures were exposed to vehicle control (PBS) or 10 ug/ml of monoclonal antibodies against mouse CD47, CD24, or the combination for 2 hours. Phagocytosis is represented as CD45+ macrophages that had engulfed CFSE+ KPCA.C cells as a percentage of the total macrophage population. D, Quantification of phagocytosis for cell lines in A . Cell lines are organized based on expression levels of each surface marker. Data represent mean ± SD of 3 technical replicates. E, Correlation of cell surface expression levels of CD47 and CD24 compared to phagocytosis upon treatment with the corresponding antibodies for each cell line. Data points depict mean ± SD from 3 replicates for each experiment. Correlation was assessed by simple linear regression. F, Representative microscopy images of GFP+ KPCA.C cells when co-cultured with primary mouse macrophages upon treatment with vehicle control (PBS), 10 ug/mL anti-CD47, 10 ug/mL anti-CD24, or the combination for 6.5 days. Top row depicts raw images of GFP+ fluorescence. Bottom row depicts purple GFP+ mask for above images used for quantification of cancer cell growth. Scale bar, 800 µm. G, Quantification of fluorescent well area from co-culture experiments for multiple cell lines after 6.5 days, organized by surface expression of CD24. Cancer cells were quantified by either green (KPCA.C, 3LL ΔNRAS, MC38) or red (238N1) fluorescent area based on their fluorophore expression. Data and means shown from one (3LL ΔNRAS, MC38) or two (238N1, KPCA.C) independent experiments with 3 technical replicates per experiment. D,G, statistical significance ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 determined by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Bioprocessing, Marker, Microscopy, Fluorescence, Co-Culture Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Representative histograms demonstrating cell surface expression of CD47 and CD24 on knockouts of KPCA.C and knockdowns of 238N1 by flow cytometry. B, Representative gating of phagocytic APC CD45+ mouse macrophages when co-cultured with the indicated CFSE+ KPCA.C knockouts treated with vehicle control (PBS) for 2 hours. Phagocytic macrophages are calculated as CD45+ cells that have engulfed CFSE+ cancer cells after 2 hours as a percent of all macrophages. C,D, Quantification of phagocytosis as a percentage of the maximum phagocytic response of macrophages using KPCA.C knockout cells ( C ) or 238N1 knockdown cells ( D ) treated with vehicle control (PBS), anti-mouse CD47 antibody, anti-mouse CD24 antibody, or the combination. Data represents mean ± SD of 3 technical replicates. E,F, Quantification of fluorescent well area as a measure of GFP+ KPCA.C knockout cells ( E ) or mCherry+ 238N1 knockdown cells ( F ) growth after co-culture with primary mouse macrophages and the indicated antibodies on day 6.5. Data represents mean ± SD from two independent experiments of 3 technical replicates each. G,H, Quantification of phagocytosis using CFSE+ MC38 ( G ) or 3LL ΔNRAS ( H ) cancer cells that overexpress CD24 after co-culture with primary mouse macrophages and the indicated antibodies. Data represent mean ± SD from 3 individual experiments each containing 3 technical replicates. I, Quantification of phagocytosis using StayGold+ KPCA.C cancer cells treated with vehicle control (PBS), or anti-mouse CD24 antibody, in the absence or presence of FcR blocking reagents (Fc1, anti-mouse Truestain clone 93; Fc2, anti-mouse CD16/CD32 clone 2.4G2). Data represents mean ± SD of 3 technical replicates. ( C-H ) ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Knock-Out, Knockdown, Co-Culture Assay, Blocking Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: Results of scRNA-seq of sorted CD45+ immune cells from experiments using CD24 or CD47 knockout tumors. ( A,C,E ) Comparison of CD47- tumors (KPCA.C CD47 knockout, 238N1 CD47 knockout) to wild-type tumors (KPCA.C control, 238N1 control). A, Relative frequencies of immune cells from CD47- versus wild-type tumors. C, UMAP showing identified cell clusters. E, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways. ( B,D,F ) Comparison of CD24- tumors (KPCA.C CD24 knockout, 238N1 CD24 knockout) to wild-type tumors (KPCA.C control, 238N1 control). B, Relative frequencies of immune cells from CD24- versus wild-type tumors. D, UMAP showing identified cell clusters. F, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Knock-Out, Comparison, Control

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Diagram showing process for high-throughput development and functional evaluation of bispecific antibodies targeting macrophage immune checkpoints. Antibody sequences were transformed into scFvs and cloned into a knob-into-hole format using a human IgG1 Fc. Constructs targeting macrophage immune checkpoints (CD47, CD24, SIRPa, PD-1) were cloned into knob formats and crossed with tumor-binding constructs in a hole format. Bispecific antibodies (n = 77) were expressed in Expi293F cells and used for downstream biochemical and functional analysis. B, Growth of StayGold+ DLD-1 cells in co-culture with human macrophages and each bispecific antibody. Each curve represents the mean for an individual bispecific antibody from 4 replicates. Black curve with hashed lines represents mean and 95% CI of control wells . C, Anti-tumor efficacy of bispecific antibodies at approximately t = 6.5 days as evaluated by macrophage checkpoint category. *p<0.05, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. D-F, Growth curves for each of the WTa2d1 constructs ( D ), CD24-3 constructs ( E ), or CV1 constructs ( F ). G, Representative whole-well imaging of co-cultures treated with different bispecific antibodies at approximately t = 6.5 day. Green signal depicts growth of StayGold+ DLD-1 cells. Rows contain different macrophage checkpoint arms, while columns contain different tumor-binding arms. H, Scatter plot showing binding of each bispecific antibody to human neutrophils versus red blood cells. I, Representative histograms showing binding of the indicated bispecific antibodies to human neutrophils and red blood cells.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: High Throughput Screening Assay, Functional Assay, Transformation Assay, Clone Assay, Construct, Binding Assay, Co-Culture Assay, Control, Imaging

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Surface CD47 (A) and ecto-CRT protein expression (B) shown as geometric MFI in a panel of six different NSCLC cell lines. Each histogram represents the mean (± SD) of three to five independent experiments. Comparisons made by ANOVA with Fisher's post hoc multiple comparison analysis. ### p < 0.03, ## p < 0.01, # p < 0.0005. Below each histogram, a matrix table where all p values resulting from post hoc analysis are reported. Expression levels of CD47 (C) and CRT mRNA (D) in 226 untreated primary NSCL adenocarcinomas (GEO accession number GSE31210 ). Middle lines in box plots represent the medians and whiskers represent 5–95% CI ( ### p < 0.03, Kruskal-Wallis test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Modulation by gefitinib of surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Flow cytometric profiles of surface CD47 (A) and ecto-CRT expression (B) on DMSO-treated (CTRL, gray lines) and gefitinib-treated (GEF, red lines) NSCLC cells. Histograms show the mean (± SD) of fold changes of CD47 (C) and ecto-CRT (D) geometric MFI, relative to DMSO-treated controls ( N = 3–5, * p < 0.05, ** p < 0.01 paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Gefitinib-induced CD47 down-regulation promotes tumor cell phagocytosis by dendritic cells. Representative flow cytometric analyses and mean ± SD ( N = 4 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells (see Methods) against PC9 (A,B) , HCC827 (C,D) , and H1975 cells (E,F) treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Cancer cells exposed to the drug for 48 h were labeled with DiO tracer and then co-cultured with dendritic cells for 2 h at a 1:1 ratio. Phagocytosis assays were also run at 4°C as controls. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (* p < 0.05, n.s., not significant, paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Activity Assay, Derivative Assay, Labeling, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Blocking of CD47 on tumor cells induces phagocytosis by dendritic cells. Dendritic cells were co-cultured with DiO tracer-labeled HCC827 (A) and H1975 (B) cancer cells in the presence of IgG isotype control or anti-CD47 mAb as indicated. Shown is the mean (± SD, N = 3 independent healthy donors) percentage increase of CD11c/DiO tracer double positive cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( # p < 0.05, ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Blocking Assay, Cell Culture, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Expression levels of surface CD47 increase in cancer cells acquiring resistance to gefitinib and inhibit tumor cell phagocytosis by dendritic cells. Surface CD47 (A) and ecto-CRT expression (B) in gefitinib-sensitive PC9 and HCC827 (gray lines) and resistant PC9GR and HCC827GR (green lines) cell lines. Representative flow cytometric histograms (left) and mean (± SD, N = 3–5) fold changes of treatment-resistant over sensitive cells (right). (C) Representative flow cytometric histogram plots (left) and mean (± SD) fold changes (right) of surface CD47 levels in resistant cell lines treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Acquisition of resistance to gefitinib abolished drug-induced CD47 down-regulation in PC9GR (* p < 0.05, ** p < 0.01, n.s., not significant, paired two-tailed Student's t -test). (D) Mean ± SD ( N = 3 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells against PC9GR cells in the absence or presence of gefitinib treatment, performed at 4°C as control and at 37°C. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (paired two-tailed Student's t -test. n.s., not significant). (E) Dendritic cells were co-cultured with gefitinib-treated, DiO tracer-labeled PC9GR cells in the presence of IgG isotype control or anti-CD47 mAb. Shown is the mean ± SD ( N = 3 independent healthy donors) percent change of CD11c + /DiO + tracer double positive dendritic cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Two Tailed Test, Activity Assay, Derivative Assay, Control, Cell Culture, Labeling

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 regulates mucosal wound healing in vivo. Utilizing a miniature video endoscope and biopsy scissors, 5–7 wounds were created in the dorsal aspect of the descending colon mucosa of anesthetized mice. a Digital measurement of wound surface area at 24 and 72 h post wounding revealed a striking impairment in wound closure in Cd47 −/− mice. Points represent the mean value within all wounds from individual mice. Data are representative of three independent experiments with five mice per group and are expressed as means ± SEM. *** p < 0.001; two-tailed Student’s t test. b In total, 10 µg of control antibody (IgG) or anti-CD47 antibody (miap301 or miap410) were injected into wound beds of wounds created 24 h previously in C57Bl/6 mice, resulting in substantial reduction of wound closure upon blockade of CD47. c Mice treated locally or systemically with anti-CD47 monoclonal antibodies miap301 or miap410 experienced less wound area reduction in comparison with IgG-treated controls. Points represent mean value within all wounds from an individual mouse. Data are representative of two independent experiments with five mice per group. Date are means ± SEM. *** p < 0.001; one-way ANOVA. Scale bars: 50 mm. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: In Vivo, Two Tailed Test, Control, Injection, Bioprocessing, Comparison

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: Loss of CD47 in IEC does not induce immune-mediated mucosal damage. a – c Naive Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice housed in pathogen-free conditions were analyzed for intestinal epithelial CD47 expression. Cd47 ERΔIEC mice were treated with tamoxifen to induce acute CD47 deletion in intestinal epithelial cells, and analyzed 2 weeks later. a Tissue sections from naive Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice were stained with anti-CD47 antibodies (green) with DAPI counterstain (blue). CD47 expression is absent in the epithelium but retained in the lamina propria and submucosa. Scale bars = 100 μm upper panels, 50 μm lower panels. b IECs were isolated from the terminal ileum of naive mice. Protein lysates were analyzed by SDS–PAGE and immunoblot for CD47. c IECs were isolated from Cd47 ERΔIEC mice treated with vehicle (corn oil) or tamoxifen, and analyzed as in b . Results are representative of three independent experiments with 3–5 mice per group. d Paraffin-embedded colon tissue sections from Cd47 ΔIEC mice were stained with Hematoxylin and Eosin counterstain for histological examination. Gross mucosal architecture is intact in the absence of epithelial CD47 expression. Scale bars = 50 μm upper panels, 100 μm lower panels. e Colon tissue digests from naive Cd47 ΔIEC mice were stained and analyzed by flow cytometry, showing no significant differences in major lamina propria immune cell populations. Points represent individual samples each containing two mice ( n = 4 mice per group). Data are means ± SEM and are representative of three independent experiments. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Expressing, Staining, Isolation, SDS Page, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: IEC-specific deletion of CD47 results in impaired mucosal healing. Utilizing a miniature video endoscope and biopsy scissors, 5–7 wounds were created in the dorsal aspect of the descending colon mucosa of anesthetized mice. Cd47 ERΔIEC mice were wounded 2 weeks after tamoxifen treatment. a Digital measurement of wound surface area at 24 and 72 h post wounding revealed a striking impairment in wound closure in Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice. Points represent mean value within all wounds from an individual mouse. Data are representative of two independent experiments with 5–6 mice per group. Data are means ± SEM. *** p < 0.001; one-way ANOVA. b Tissue sections taken from day 3 wounds were stained with the epithelial-specific marker E-Cadherin (green), plus DAPI counterstain (blue). Re-epithelialization of the wound is disorganized in the absence of CD47 ( Cd47 ΔIEC ) compared with control Cd47 f/f . c Tissue sections taken from day 3 wounds were stained with the epithelial-specific marker E-Cadherin (green), brush border protein Villin (magenta), and DAPI (blue). Insets of epithelial cells on top of the wound bed show polarized wound-associated epithelial cells (WAE) expressing Villin (arrows) in Cd47 f/f wounds while cells are not polarized in Cd47 ΔIEC wounds. Scale bars = 100 μm. d Ki67 staining of frozen sections of wounded colon mucosa 3 days post-wounding (red) revealed similar proliferation rates in crypt epithelial cells immediately adjacent to wounds in the absence of epithelial CD47. Sections were counterstained with E-Cadherin (green) and DAPI (blue). Scale bars = 50 μm. Points represent the average number of Ki67-positive cells for four crypts adjacent to wounds for each individual mouse. Data are means ± SEM and are representative of two independent experiments with 4–6 mice per group. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Staining, Marker, Control, Expressing

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: Loss of CD47 in IEC results in impaired recovery from DSS-induced colitis. Age- and sex-matched Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice were treated with three consecutive cycles of 2.5% DSS in drinking water for either 4 days ( Cd47 ΔIEC ) or 3 days ( Cd47 ERΔIEC ), followed by 5 days of water recovery. Cd47 ERΔIEC mice were treated with DSS 2 weeks after tamoxifen treatment. a – b Disease activity index scores are represented as an average of scores 0–4 for percent weight loss, stool consistency, and presence of blood in stools. Cyclical treatment of a Cd47 ΔIEC and b tamoxifen-treated Cd47 ERΔIEC mice with 2.5% DSS in drinking water, followed by a plain water recovery period, induced greater DAI scores in the absence of epithelial CD47. Data are representative of two independent experiments with 5–6 mice per group and are expressed as means ± SEM. a * p = 0.02, b * p = 0.036 by two-way ANOVA. c Histological scoring of hematoxylin and eosin (H&E)-stained tissue sections of colonic mucosa: percentage of injury/ulceration represents a ratio of the length of injured/ulcerated areas (≥ 50% crypt loss) relative to the entire colon length, as assessed in Swiss roll mounts of the entire colon. Results indicate greater damage in the absence of epithelial CD47. Points represent individual mice. Data are representative of two independent experiments with 5–6 mice per group and are expressed as means ± SEM. Significance determined by two-tailed Student’s t test. * p = 0.016, *** p = 0.001. d , e Representative H&E staining of colon tissue sections after three cycles of DSS/water revealed extensive crypt destruction in distal colon of Cd47 ΔIEC and Cd47 ERΔIEC mice. Large areas of ulcerated mucosa with granulocytic infiltrates were present in the mid colon in Cd47 ΔIEC and Cd47 ERΔIEC mice, in comparison with littermate controls. Scale bars = 100 μm upper panels, 300 μm lower panels. Results are representative of at least two independent experiments with 5–6 mice per group. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Activity Assay, Staining, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 is required for wound repair in cultures of primary epithelial monolayers. a Primary epithelial cell monolayers derived from CD47-expressing (CD47( + )) or CD47-deficient (CD47(−)) murine enteroids were scratch-wounded and monitored for closure. CD47(−) epithelial monolayers showed significant impairment in reduction of scratch-wound surface area at 24 h post scratch. Edges of scratch wounds are indicated by dashed lines. Scale bars = 50 μm. b Primary epithelial cell monolayers derived from human stem cell-derived colonoids were scratch-wounded and treated with 10 µg/ml of either IgG control antibody, function-blocking anti-CD47 antibody (clone B6H12), or non-blocking anti-CD47 antibody (clone 2D3), resulting in the inhibition of cell migration upon blockade of CD47. a – b Results are representative of three independent experiments with three replicates per treatment group. Data are means ± SEM. Significance determined by two-way ANOVA, ** p ≤ 0.01, *** p ≤ 0.001. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Derivative Assay, Expressing, Control, Blocking Assay, Inhibition, Migration

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 associates with β1 integrin and promotes focal adhesion formation. a In situ proximity ligation assay utilizing antibodies against CD47 and β1 integrin indicating close association between CD47 and β1 integrin in the colonic epithelium. Positive PLA signals are shown in green, beta-catenin in magenta and DAPI/nuclei in blue. Crypts are indicated by dashed lines. Arrowheads and asterisks (*) indicate positive PLA signals on IECs and immune cells in the lamina propria, respectively. Strong PLA signals are detected in IECs that are mainly concentrated at the base of the crypts in Cd47 f/f colon in contrast to very few PLA signals are observed in crypts IECs in Cd47 ΔIEC colons. Scale bars = 20 μm. The specificity of the PLA signals was evaluated in Supplementary figure demonstrating no PLA signals in Cd47 −/− colons. b Whole-cell lysates from freshly isolated intestinal epithelial cells from Cd47 f/f and Cd47 ΔIEC mice were subjected to SDS–PAGE and immunoblot for signaling molecules downstream of β1 integrin-dependent cell adhesion, revealing decreased β1 integrin protein expression and reduced phosphorylation of Src Y416 , FAK Y397/Y861 , and p130CAS Y410 in cells from Cd47 ΔIEC mice. Results are representative of three independent experiments. c Murine enteroid-derived primary epithelial cell monolayers were scratch-wounded and harvested at the indicated time points, then analyzed by SDS–PAGE and immunoblot. CD47-deficient epithelial cells show a reduction in phosphorylated Src Y416 , FAK Y397/Y861 , and p130CAS Y410 upon wounding, whereas maintaining reduced β1 integrin protein baseline expression. d Epithelial cells immediately adjacent to wounds from Cd47 f/f and Cd47 ΔIEC mice were imaged by confocal microscopy, showing disrupted basal co-staining for phosphorylated FAK Y861 and β1 integrin (apical surface indicated by dashed line). Arrows indicate reduced colocalization of phospho-FAK Y861 and β1 integrin in the absence of CD47 expression. Scale bars = 10 μm. Results are representative of three independent experiments with three mice per treatment group. e Lamellipodia of CD47(−) cells exhibited fewer phosphorylated FAK Y861 -positive focal adhesions in comparison with CD47-expressing cells (insets). Scale bars = 10 μm. Results are representative of three independent experiments with two independently derived enteroid culture lines. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: In Situ, Proximity Ligation Assay, Isolation, SDS Page, Western Blot, Expressing, Phospho-proteomics, Derivative Assay, Confocal Microscopy, Staining, Comparison

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 regulates thrombospondin-1, TGF-β1, and collagen deposition after injury. a Whole cell lysates from freshly isolated intestinal epithelial cells from Cd47 f/f and Cd47 ΔIEC mice were subjected to SDS–PAGE and immunoblot for thrombospondin-1/TSP-1, TGF-β1, and phosphorylated SMAD2 and SMAD3. Results are representative of three independent experiments. b Representative Masson’s trichrome staining of wounds beds and chronic DSS-colitis colons from Cd47 f/f and Cd47 ΔIEC mice. Scale bars = 50 μm. Results representative of three independent experiments with 5–7 mice per group. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Isolation, SDS Page, Western Blot, Staining

Journal: Journal of nanobiotechnology

Article Title: Thrombolytic therapy based on lyophilized platelet-derived nanocarriers for ischemic stroke.

doi: 10.1186/s12951-023-02206-5

Figure Lengend Snippet: Fig. 1 A Schematic representation of the preparation of rtPA-loaded CSM derived from platelets. B Representative mean hydrodynamic diameter of CSM and CSM@rtPA before and after lyophilization (CSM@rtPA/L) process measured by DLS. Data represent mean ± SEM (n = 3, independent samples). C Representative mean diameter of CSM and CSM@rtPA before and after lyophilization process (CSM@rtPA/L) measured by NTA. Data represent mean ± SEM (n = 3, independent samples). D Representative STEM-in SEM images of CSM@rtPA negatively stained with uranyl acetate. Scale bars: 200 nm. E Loading capacity of rtPA encapsulated in CSM samples. Data represent mean ± SEM (n = 3, independent samples). F Representative density plots and quantitative analysis of CSM and CSM@rtPA measured by FC. Scatter density plots of Green fluorescence signal (Green-B channel, rtPA channel) versus Red fluorescence signal (Red-R channel, CellMask DeepRed channel) for CSM, CSM@rtPA, and CSM@rtPA sample after labeling with CellMask Deep Red for lipid staining. Mean fluorescence intensities (MFI) for Green-B channel (D) and Red-R channel. Data represent mean ± SEM (n = 3, independent samples). G. Schematic representation of platelets and platelet-derived CSM surface proteins studied by APC-fluorescently labeled antibodies: anti-hCD47 Ab and anti-hCD42b/GPlba Ab. In vitro Ab binding to platelets and CSM@rtPA before and after lyophilization process. Representative MFI histogram of Red-R channel (APC signal) for platelets, CSM@rtPA and CSM@rtPA/L samples after incubation with APC-anti-CD47 and APC-anti-CD42b/GPIbα antibodies

Article Snippet: Then, fluorescently antibodies, human anti-CD47 APC conjugated Antibody (FAB4670A, R&D Systems) and human antiCD42b/GPlbα APC conjugated Antibody (FAB4067A, R&D systems) were incubated with platelets and with CSMs (10 μL of antibody stock solution /106 cells) and the samples were analyzed by flow cytometer.

Techniques: Derivative Assay, Lyophilization, Staining, Fluorescence, Labeling, In Vitro, Binding Assay, Incubation

Journal: Oncoimmunology

Article Title: IRE1α overexpression in malignant cells limits tumor progression by inducing an anti-cancer immune response

doi: 10.1080/2162402X.2022.2116844

Figure Lengend Snippet:

Article Snippet: PE-Vio770 anti-CD47 , REAfinity, Miltenyi Biotec , 130–103-105, RRID:AB_2659751.

Techniques: