Journal: Nature Communications

Article Title: Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

doi: 10.1038/s41467-020-18245-7

Figure Lengend Snippet: a Co-expression of CD47 and HER2 across multiple human cancers (red) compared to corresponding normal tissues (blue) obtained from GEPIA database; expression of CD47 and HER2 in 1085 BC patients and 291 normal tissues are shown in the right. T tumors, N normal tissues. * P < 0.05. b Expression of CD47 preferring in HER2-expressing BC cells (MCF7, MCF7/C6, SKBR3, and BT474) compared to HER2-negative MDA-MB-231 and BT549 cells. c Immunoblotting of HER2 and CD47 in tumors from diagnosed HER2 + and HER2 − BC patients. d Left, representative images scored as low, moderate, and high CD47 staining. Right, numbers of patients with low, medium, or high IHC staining of CD47 grouped by HER2 positive or negative status (total HER2 + tumors n = 18; total HER2 − tumors n = 18). e Left, representative IHC of CD47 and HER2 in paired primary and recurrent samples from the same patients. Right, numbers of patients with low, medium, or high IHC staining of CD47 grouped by primary or recurrent status (total primary tumors n = 18; total recurrent tumors n = 18). f Probability of recurrence-free (RFS) and g distant metastasis-free survival (DMFS) of BC patients of all subtypes stratified according to HER2 CD47 signature expression within HER2 strata from Breast Cancer Meta-base: 10 cohorts 22k genes database generated by SurvExpress ( http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp ) from the HER2 probe 210930_s_at combined CD47 probe 211075_s_at. Statistical significance was analyzed by log-rank test.

Article Snippet: Hybridoma for monoclonal mouse anti-human CD47 antibody (B6H12.2), IgG2b were obtained from ATCC.

Techniques: Expressing, Western Blot, Staining, Immunohistochemistry, Generated

Journal: Nature Communications

Article Title: Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

doi: 10.1038/s41467-020-18245-7

Figure Lengend Snippet: Expressions of HER2 and CD47 in radioresistant MDA-MB-231/C5 cells ( a ) in radioresistant MCF7/C6 cells ( b ) both were derived from surviving clones after FIR. c Expression of HER2 and CD47 in syngeneic mouse 4T1 breast tumors 16 h after local RT with single-dose radiation or FIR (pooled samples from six tumors). d Enhanced CD47 + /HER2 + population in MCF7/C6 cells compared to parental MCF7 cells measured by flow cytometry ( n = 3; * P < 0.05, *** P < 0.001, **** P < 0.0001). e Induction of CD47 by radiation in an array of human cancer cells (breast cancer MCF-7, BT474, SKBR3, glioblastoma U251, and colorectal carcinoma HCT116 cells) measured by immunoblotting 16 h after radiation (5 Gy). f Induction of CD47 expression in xenograft tumors (duplicated samples from each tumor group) following RT with fractionated doses (FIR, 2 Gy × 5).

Article Snippet: Hybridoma for monoclonal mouse anti-human CD47 antibody (B6H12.2), IgG2b were obtained from ATCC.

Techniques: Derivative Assay, Clone Assay, Expressing, Flow Cytometry, Western Blot

Journal: Nature Communications

Article Title: Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

doi: 10.1038/s41467-020-18245-7

Figure Lengend Snippet: Radiation enhanced CD47 + cell populations were reduced by inhibition of HER2 in MCF7/C6 ( a ) and in SKBR3 cells ( b ) treated with Lapatinib (10 µM, 72 h) in the presence or absence of IR (5 Gy) ( n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001). c Immunoblotting of CD47 in HER2 + RD-BCSCs treated with PBS or Herceptin (10 µg/ml for 5 days refreshed on 2nd and 4th day) followed by sham or 5 Gy IR (HER2 − RD-BCSCs included as a control) . d CD47 + populations detected by flow cytometry in HER2 + RD-BCSCs and HER2 + RD-BCSCs with CRISPR-mediated knockout of HER2 (HER2 −/− ), n = 3; ** P < 0.01. e Basal and radiation-induced CD47 expression was absent in HER2 + RD-BCSCs with CRISPR-mediated knockout of HER2 (HER2 −/− ). f NF-κB luciferase reporter activity in radioresistant MCF/C6 cells and MCF/C6 cells with HER2 −/− , n = 3, *** P < 0.001. g CD47 promoter-controlled luciferase reporter activity containing wild-type or mutant NF-κB-binding motif in MCF7/C6 cells treated with TNFα ( n = 3, *** P < 0.001). h CD47 promoter activity with wild-type or mutant NF-κB-binding motif measured in control and irradiated MCF7/C6 cells in the presence or absence of NF-κB inhibitor IMD-0354 (2 μM, 5 h; n = 3, ** P < 0.01, *** P < 0.001). i Representative images of immunofluorescence of CD47 in irradiated MCF7 and MCF7/C6 cells pretreated with IMD-0354 (IMD); CD47 was visualized by confocal microscope 16 h after IR. Scale bar, 25 µm. j Immunoblotting of CD47 and HER2 in IR-treated MCF7/C6 cells with or without IMD-0354. k ChIP-qPCR assay for NF-κB recruitment in human CD47 promoter in MCF7/C6 cells irradiated (5 Gy) or treated with 10 ng/ml TNF-α. Chromatin precipitation was conducted with anti-p65 where anti-c-Rel served as a positive control in IκB-α promoter, IgG served as a negative control. l Reduced HER2-expressing populations in MCF7/C6 cells with CRISPR/Cas9-mediated knockout of CD47.

Article Snippet: Hybridoma for monoclonal mouse anti-human CD47 antibody (B6H12.2), IgG2b were obtained from ATCC.

Techniques: Inhibition, Western Blot, Control, Flow Cytometry, CRISPR, Knock-Out, Expressing, Luciferase, Activity Assay, Mutagenesis, Binding Assay, Irradiation, Immunofluorescence, Microscopy, ChIP-qPCR, Positive Control, Negative Control

Journal: Nature Communications

Article Title: Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

doi: 10.1038/s41467-020-18245-7

Figure Lengend Snippet: a Macrophage-mediated phagocytosis was enhanced in both MCF7 and MCF7/C6 cells by CD47 antibody detected by flow cytometry analysis with THP1-derived mature macrophages stained with DIO and incubated with 3 × 10 4 DDAO-labeled tumor cells at a ratio of 5:1 for 2 h at 37 °C. For anti-CD47 treatment, tumor cells were pre-incubated with 10 µg/ml of CD47 antibody (B6H12. 2) for 1 h prior to incubation with macrophages ( n = 3, ** P < 0.01). Mammosphere formation ( b , n = 3; ** P < 0.01), transwell invasion ( c , au arbitrary units; n = 3; * P < 0.05), and gap-filling assay ( d , n = 3; ** P < 0.01) of MCF7/C6 cells with or without anti-CD47 treatment (no treatment and IgG were used as controls). e , f Enhanced macrophage-mediated phagocytosis on MCF7/C6 cells treated with Lapatinib, Herceptin, B6H12.2, or combination of Herceptin and B6H12. 2 in MCF7/C6 cells ( n = 3, * P < 0.05, ** P < 0.01). g Clonogenic survival of MCF7/C6 cells received radiation with different antibody combinations (IgG or B6H12. 2, 10 µg/ml for overnight; Herceptin, 10 µM for 72 h; IR, 5 Gy) was calculated by normalizing to the clonogenic fractions of control cells received 0 Gy IR ( n = 3, * P < 0.05, ** P < 0.01). h Lack of targeted gene expression in CD47 −/− or HER2 −/− in MCF7/C6 cells. i Enhanced macrophage phagocytosis on CD47 −/− or HER2 −/− MCF7/C6 cells (* P < 0.05). j Immunoblotting of CD47 and HER2 in MCF7/C6 cells with CD47 −/− /HER2 −/− status. k Macrophage phagocytosis of CD47 −/− /HER2 −/− MCF7/C6 cells (* P < 0.05). l Clonogenic radiosensitivity of MCF7/C6 cells with CD47 −/− , HER2 −/− , or CD47 −/− /HER2 −/− status calculated by normalizing to the clonogenic fractions of control cells received 0 Gy IR ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Hybridoma for monoclonal mouse anti-human CD47 antibody (B6H12.2), IgG2b were obtained from ATCC.

Techniques: Flow Cytometry, Derivative Assay, Staining, Incubation, Labeling, Control, Targeted Gene Expression, Western Blot

Journal: Nature Communications

Article Title: Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

doi: 10.1038/s41467-020-18245-7

Figure Lengend Snippet: a Growth rate of mouse breast tumors generated with radioresistant 4T1/C2 cells with CD47 −/− , HER2 −/− , or CD47 −/− /HER2 −/− . b Representative images of tumors at the end of experiments. c Repeated experiment of a with a single dose IR (5 Gy) at day 9 and representative images of the tumor at the end of experiments shown in d ; in a and c , n = 6, mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001. e Left, representative IHC images of CD11b + macrophages in CD47 −/− , HER2 −/− , CD47 −/− /HER2 −/− tumors; scale bar = 100 µm; right, quantitation of CD11b + macrophages ( n = 4, * P < 0.05, *** P < 0.001).

Article Snippet: Hybridoma for monoclonal mouse anti-human CD47 antibody (B6H12.2), IgG2b were obtained from ATCC.

Techniques: Generated, Quantitation Assay

Journal: Nature Communications

Article Title: Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

doi: 10.1038/s41467-020-18245-7

Figure Lengend Snippet: Tumor volumes ( a ), tumor weights at end of experiments ( b ), and representative images ( c ) were measured with mouse syngeneic breast tumors generated with radioresistant 4T1/C2 cells and treated with intratumoral injection of PBS, IgG, Herceptin (5 mg/kg), anti-CD47 antibody (5 mg/kg), or a combination both antibodies every other day starting at day 10 with radiation delivered locally to tumors of all groups with 5 Gy at days 10 and 11 ( n = 6, mean ± SEM, ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). d Above, HE staining of 4T1/C2 tumors treated with radiation combined with indicated antibodies. Bottom, representative IHC images of CD11b + macrophages in tumors; scale bar = 100 µm. e Representative images of infiltrated macrophage and macrophage-mediated phagocytosis in the syngeneic mouse orthotopic BC models repeated as in a with GFP-labeled 4T1 tumors detected with indicated antibodies; green, tumor cells; red, infiltrated macrophages; blue, cell nucleus stained with DAPI; yellow (indicated with arrows), macrophage-mediated phagocytosis with tumor cells. f Quantitation of relative richness of infiltrated CD11b + macrophages. g Quantitation of macrophages-mediated phagocytosis with tumor cells counted in multiple areas in the high-power fields marked with yellow arrows (Image Pro Plus 6.0; b , c , * P < 0.05, **** P < 0.0001; ns not significant).

Article Snippet: Hybridoma for monoclonal mouse anti-human CD47 antibody (B6H12.2), IgG2b were obtained from ATCC.

Techniques: Generated, Injection, Staining, Labeling, Quantitation Assay

Journal: Nature Communications

Article Title: Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

doi: 10.1038/s41467-020-18245-7

Figure Lengend Snippet: The radioresistant breast cancer cells that are enriched with breast cancer stem cells can not only survive radiation but also enhance their immune-escaping ability via CD47-mediated anti-phagocytosis. CD47 transcription is regulated via HER2–NF-κB axis leading to the enhancement of both receptors on the cell surface. Thus, HER2-promoted intrinsic proliferation is synergized with the CD47-initiated extrinsic anti-phagocytosis; a dual blockade of these two receptors may eliminate the radioresistant breast cancer cells in breast cancer radiotherapy.

Article Snippet: Hybridoma for monoclonal mouse anti-human CD47 antibody (B6H12.2), IgG2b were obtained from ATCC.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Expression of ARID1A and CD47 and their relationship in GC tissues. (A,B) Representative IHC images of ARID1A staining, ARID1A-preserved expression (A) , ARID1A-loss expression (B) . (C,D) Representative IHC images of CD47 staining, CD47 high expression (C) , CD47 low expression (D) . (E) Relationship between loss/preserved expression of ARID1A and CD47 expression ( n = 154). (F) Relationship between mutated/wild-type expression of ARD1A and CD47 expression (data from TCGA, n = 375). * p < 0.05, *** p < 0.001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: The correlation between ARID1A status and CD47 expression and clinicopathological characteristics.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Loss of ARID1A increases CD47 expression. (A,B) Protein and mRNA expression levels of ARID1A and CD47 among various GC cell lines. (C–E) Knockdown of ARID1A significantly increased total and membranous CD47 protein levels, and CD47 mRNA level measured by western blot, flow cytometry and qRT-PCR in GC cell lines. (F) Schematic illustration of the relative positions of qPCR probes to putative CD47 promoter region for ChIP-qPCR experiments. (G) ARID1A binding to the promoter region of CD47 was determined by ChIP-qPCR in BGC-823 and MKN28 cells, respectively. *** p < 0.001, **** p < 0.0001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Western Blot, Flow Cytometry, Quantitative RT-PCR, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Kaplan–Meier curves under various conditions. (A,B) Survival curves according to ARID1A status or CD47 expression ( n = 154). (C) Survival curves according to ARID1A status combined with CD47 expression level ( n = 154). (D–G) Survival curves according to ARID1A status or CD47 expression with HER2 positive or negative.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Characterization of tumor-infiltrating immune cells based on ARID1A and CD47 expression. (A) Estimation of tumor-infiltrating immune cells in different subgroups stratified by ARID1A status and CD47 expression. (B) Multiplex immunofluorescent images illustrate GC tissues with ARID1A loss CD47 high or ARID1A preserved CD47 low expression that express CD11c, CD163, and Foxp3. (C) Quantitative analysis of the densities of immune markers between ARID1A loss CD47 high and ARID1A preserved CD47 low GC tissues. Error bars represent mean with SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Combining radiation therapy with anti-CD47 mAb treatment enhances in vitro phagocytosis of human DIPG and mouse glioma cell lines. Human patient-derived DMG cell lines (BT-245, SU-DIPGXVII, and SU-DIPGXXV) were exposed to either 0 or 4 Gy×3 and incubated with human peripheral blood-derived macrophages in the presence of anti-CD47 mAb, HU5F9-G4. Flow cytometry ( A , C , and E ) as well as histogram ( B , D , and F ) plots show that combining fractionated irradiation and anti-CD47 antibody treatment increases the phagocytosis of DIPG/DMG cells by macrophages compared with individual treatments alone. ( G–J ) Mouse glioblastoma cell lines (SB28 and CT2A) were exposed to either 0 or 4 Gy×3 and incubated with mouse bone marrow-derived macrophages in the presence of anti-CD47 mAb, MIAP301. Data shown are consistent with two independent experiments (n=3) and are shown as mean+SD. Unpaired Student’s t-test. *p<0.05, **p<0.01 and ***p<0.0001. One-way analysis of variance, ****p<0.0001 (BT245), ***p<0.001 (DIPGXVII) and ****p<0.0001 (DIPGXXV). ***p<0.001 (CT2A) and **p<0.001 (SB28). DMG, diffuse midline glioma; mAb, monoclonal antibody.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: In Vitro, Derivative Assay, Incubation, Flow Cytometry, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Single-cell RNA sequencing reveals the enrichment of distinct macrophage subsets following co-culture with diffuse midline glioma cells pretreated with either control, RT, anti-CD47 therapy, or combination of RT and anti-CD47 therapy. ( A ) Schematic diagram of the workflow used for in vitro phagocytosis and single-cell RNA sequencing. Briefly, BT245 cells were irradiated (4Gy×3) treated for three consecutive days and treated with either PBS or anti-CD47 mAb for 30 min at 37°C and co-cultured with PBMC-derived macrophages for 24 hours. BT245 cells not exposed to radiation and treated with PBS served as controls. Macrophages that either phagocytose (“eaters”) or do not phagocytose (“non-eaters”) tumor cells were sorted using flow cytometry and subjected to single-cell RNA-sequencing. ( B ) UMAP projection displaying 11 distinct cell clusters from the eater’s cohort. Each dotted line and arrow indicate the identity of that specific cell cluster. ( C–D ) UMAP plots and proportion of cells in each cluster from four treatment groups: control, 4 Gy×3, anti-CD47, or 4 Gy×3+anti-CD47. Dotted lines indicate the expansion/enrichment of distinct macrophage clusters in that treatment condition. Note the expansion/enrichment of two distinct cell clusters (1 and 3) in the combination treatment. ( E ) Heatmap of marker genes in each cell cluster. Representative genes with higher gene expression for each cluster are outlined on the left. Bubble plots demonstrating expression of marker genes associated with antigen presenting ( F ), inflammatory ( G ), M1-like ( H ), M2-like ( I ), proliferation ( J ), and tumor cell signature ( K ) by the various cell clusters. The dotted box indicates the cell clusters with higher average marker gene expression. The size of the bubble dot is proportional to the percentage of cells in a cluster expressing the marker gene and the color intensity is proportional to average scaled marker gene expression within a cluster. mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; RT, radiotherapy; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: RNA Sequencing, Co-Culture Assay, Control, In Vitro, Irradiation, Cell Culture, Derivative Assay, Flow Cytometry, Marker, Gene Expression, Expressing, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Characterization of macrophages that are enriched following phagocytosis of diffuse midline glioma cells pretreated with Control, anti-CD47 therapy or RT. ( A ) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with control treated BT245 cells (cell Cluster 0) compared with macrophages that were enriched after co-culture with either anti-CD47 (cell Cluster 1) or RT (cell Cluster 3) treated BT245 cells. ( B ) Gene Ontology enrichment analysis of biological process for significantly upregulated genes between control-enriched macrophages versus anti-CD47-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. ( C ) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with anti-CD47 treated BT245 cells (cell Cluster 1) compared with macrophages that were enriched after co-culture with either control (cell Cluster 0) or RT (cell Cluster 3) treated BT245 cells. ( D ) Gene Ontology enrichment analysis of biological processes for significantly upregulated genes between anti-CD47-enriched macrophages versus control-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. ( E ) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with RT treated BT245 cells (cell Cluster 3) compared with macrophages that were enriched after co-culture with either control (cell Cluster 0) or anti-CD47 (cell Cluster 1) treated BT245 cells. ( F ) Gene Ontology enrichment analysis of biological processes for significantly upregulated genes between control-enriched macrophages versus anti-CD47-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. BP, biological process; FC, fold change; NS, not significant; RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Control, Co-Culture Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Combination of RT and anti-CD47 treatment reduces tumor burden and prolongs the survival of mice bearing BT245 xenografts compared with monotherapy. ( A ) Schematic diagram showing the experimental treatment plan followed. ( B ) Quantification of total IVIS flux values over time course. ( C ) Kaplan-Meier survival analysis of BT245 xenografts with indicated treatments, control, n=10; RT, n=9; anti-CD47, n=10; and RT+anti-CD47, n=10. The log-rank test was used to calculate statistical significance. *p<0.05, **p<0.01. ( D–F ) Bar graphs demonstrating the relative percentages of F4/80 + , CD80 + (M1-like) and CD206 + (M2-like) tumor-associated macrophages in control, RT, anti-CD47, or RT+anti-CD47 treated mice bearing BT245 xenografts. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. IP, intraperitoneal; IVIS, In Vivo Imaging System; RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Control, In Vivo Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Combination of RT and anti-CD47 treatment reduces tumor burden and prolongs the survival of mice-bearing CT-2A intracranial allografts compared with monotherapy. ( A ) Schematic diagram showing the experimental treatment plan followed. ( B ) Quantification of total IVIS flux values over time course. ( C ) Kaplan-Meier survival analysis of CT-2A allografts with indicated treatments, control, n=8; RT, n=8; anti-CD47, n=8; and RT+anti-CD47, n=8. The log-rank test was used to calculate statistical significance. *p<0.05, **p<0.01, ***p<0.001. ns, not significant. ( D–F ) Bar graphs demonstrating the relative percentages of F4/80 + , CD80 + (M1-like) and CD206 + (M2-like) tumor-associated macrophages in control, RT, anti-CD47, or RT+anti-CD47 treated mice bearing CT-2A intracranial allografts. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. IP, intraperitoneal; IVIS, In Vivo Imaging System; RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Control, In Vivo Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Validation of marker genes identified from single-cell RNA-sequencing using tumor-associated macrophages obtained from murine diffuse midline gliomas and glioblastoma intracranial allografts treated with either control, RT, anti-CD47, or RT+anti-CD47. ( A ) Dot plot indicates the average expression of CLEC7A, CD44 ( D ), and ( G ) for each cell cluster identified from single-cell RNA-sequencing. ( B–C ) Representative overlay histograms and median fluorescence intensity (MFI) values of CLEC7A, CD44 ( E–F ), and CD63 ( H–I ), expression in gliomas isolated from mice treated with either phosphate-buffered saline (control), RT, anti-CD47 therapy, or RT with anti-CD47 combination therapy. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Biomarker Discovery, Marker, RNA Sequencing, Control, Expressing, Fluorescence, Isolation, Saline

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: PLoS ONE

Article Title: Down-Regulation of Platelet Surface CD47 Expression in Escherichia coli O157:H7 Infection-Induced Thrombocytopenia

doi: 10.1371/journal.pone.0007131

Figure Lengend Snippet: A, CD47 expression on platelet surfaces measured by flow cytometry. Cells derived from CD47 −/− mice served as a negative control in CD47 immunofluorescence labeling and measurement by flow cytometry. B, CD47 expression levels on the surfaces of platelets (PLT), neutrophils (PMN), monocytes (MO), and red blood cells (RBC), respectively. Shiga toxin-negative strain 99A041 (O157:H19) was used as a control for STEC O157:H7 (strain 99G144). Data were presented as mean±SD of three independent experiments. *, p<0.05.

Article Snippet: Rat anti-mouse CD47 affinity purified mAb (clone miap301) was obtained from BD Biosciences (San Diego, CA).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Negative Control, Immunofluorescence, Labeling

Journal: PLoS ONE

Article Title: Down-Regulation of Platelet Surface CD47 Expression in Escherichia coli O157:H7 Infection-Induced Thrombocytopenia

doi: 10.1371/journal.pone.0007131

Figure Lengend Snippet: A, strain 99G144 CCF but not O157:H19 CCF decreases CD47 surface expression. B, Reduction of CD47 expression levels by strain 99G144 CCF in the presence of various antibodies at concentration of 25 µg/ml each. Data were presented as mean±SD of three independent experiments. *, p<0.05.

Article Snippet: Rat anti-mouse CD47 affinity purified mAb (clone miap301) was obtained from BD Biosciences (San Diego, CA).

Techniques: Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: Down-Regulation of Platelet Surface CD47 Expression in Escherichia coli O157:H7 Infection-Induced Thrombocytopenia

doi: 10.1371/journal.pone.0007131

Figure Lengend Snippet: Percentage of phagocytosis was calculated as the fraction of macrophages with ingested platelets of the total number of macrophages analyzed in 6–8 random selected fields per slide. Soluble CD47 extracellular domain recombinant and GST only were added at 40 µg/ml each. Data were presented as mean±SD of three independent experiments. *, p<0.05.

Article Snippet: Rat anti-mouse CD47 affinity purified mAb (clone miap301) was obtained from BD Biosciences (San Diego, CA).

Techniques: Recombinant

Journal: ACS nano

Article Title: Magnetic Metal-Organic Framework-Based Nanoplatform with Platelet Membrane Coating as a Synergistic Programmed Cell Death Protein 1 Inhibitor against Hepatocellular Carcinoma.

doi: 10.1021/acsnano.3c07885

Figure Lengend Snippet: Figure 2. Characterization of PmMN@Om&As. The DLC of PmMN@Om&As was changed by adjusting the Om and As concentration ratio in the drug-loading system (A). The size distribution (B) and zeta potential (C) of Fe3O4, MN, MN@Om&As, and PmMN@Om&As were determined by isomorphic DLS (n = 3). Images showing the morphology of MN, MN@Om&As, and PmMN@Om&As were obtained using SEM (scale bar = 200 nm) (D). Images showing the morphology and elements of MN and PmMN@Om&As were obtained using TEM (scale bar = 50 nm) (E). The protein profile of PmMN@Om&As was obtained using SDS-PAGE (F). CD47 of the PmMN@Om&As was observed by WB (G). The pore characteristics of MN and MN@Om&As were observed by the curves of nitrogen adsorption and desorption (H). The crystal structure of the sample was observed by XRD (I). The responsiveness of PmMN@Om&As to magnetic fields was tested by VSM and NdFeB permanent magnets (J). Effects of reductive and acidic environment on drug release of PmMN@Om&As (n = 3) (K). TEM images showed the effects of reductive and acidic environments on PmMN@Om&As (L). Data are presented as means ± SD.

Article Snippet: In vivo mAb antimouse CD47 and In vivo mAb antimouse PD-1 were purchased from BioXcell Co., Ltd. (New Hampshire, America).

Techniques: Concentration Assay, Zeta Potential Analyzer, SDS Page, Adsorption

Journal: ACS nano

Article Title: Magnetic Metal-Organic Framework-Based Nanoplatform with Platelet Membrane Coating as a Synergistic Programmed Cell Death Protein 1 Inhibitor against Hepatocellular Carcinoma.

doi: 10.1021/acsnano.3c07885

Figure Lengend Snippet: Figure 3. Cellular uptake and immune escape of PmMN@Om&As. The uptakes of free C6, MN@C6, and PmMN@C6 by CAFs were shown by fluorescence microscopy (A) (scale bar = 50 μm) and flow cytometry (B). The uptake mechanisms of PmMN@C6 by CAFs were shown by competitive inhibition assay (n = 3) (C). The uptakes of free C6, MN@C6, and PmMN@C6 by TILs were shown by fluorescence microscopy (D) (scale bar = 50 μm) and flow cytometry (E). The uptake mechanisms of PmMN@C6 by TILs were shown by competitive inhibition assay (n = 3) (F). The abilities of PmMN@Om&As to escape macrophage phagocytosis in vitro (G) and in vivo (H) were shown by flow cytometry. The abilities of PmMN@Om&As to escape macrophage phagocytosis in vitro (I) and in vivo (J) were inhibited by blocking CD47. Data are represented as the means ± SD *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test.

Article Snippet: In vivo mAb antimouse CD47 and In vivo mAb antimouse PD-1 were purchased from BioXcell Co., Ltd. (New Hampshire, America).

Techniques: Fluorescence, Microscopy, Flow Cytometry, Inhibition, In Vitro, In Vivo, Blocking Assay, Two Tailed Test

Journal: Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society

Article Title: CD47 Blockade Reduces Ischemia/Reperfusion Injury and Improves Survival in a Rat Liver Transplantation Model

doi: 10.1002/lt.24059

Figure Lengend Snippet: Western blot analysis of CD47 and TSP-1 expression levels (A,B) during cold ischemia and (C,D) 48 hours after OLT. A significant elevation of CD47 protein with little change in TSP-1 expression after cold ischemia was noted after 18 hours of cold ischemia in comparison with livers that were flushed and then immediately frozen. The expression of both CD47 and TSP-1 protein was reduced 48 hours after OLT in CD47mAb400-treated recipients versus the IgG group (P < 0.05).

Article Snippet: After hepatectomy, the donor liver was flushed via the portal vein with 10 mL of cold (4 °C) normal saline, which was followed by 10 mL of cold University of Wisconsin (UW) solution containing either a mouse anti-CD47 monoclonal antibody, CD47mAb400 (Vasculox, Inc., St. Louis, MO), or an isotype matched-control mouse immunoglobulin G 2a (IgG2a) at a dose of 1 μg/g liver weight.

Techniques: Western Blot, Expressing