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: Journal of Clinical Laboratory Analysis

Article Title: High‐dimensional single‐cell proteomics analysis reveals the landscape of immune cells and stem‐like cells in renal tumors

doi: 10.1002/jcla.23155

Figure Lengend Snippet: Purified antibodies about the stem‐like cell‐centric panel

Article Snippet: CD47 , 209Bi , CC2C6 , Fluidigm , 3209004B.

Techniques: Purification

Journal: Immuno-Oncology Technology

Article Title: CD47–SIRPα-targeted therapeutics: status and prospects

doi: 10.1016/j.iotech.2022.100070

Figure Lengend Snippet: Ongoing and recruiting trials of anti-CD47 and anti-SIRPα agents (by estimated study completion date, grouped by agent)

Article Snippet: TG-1801 (NI-1701) (CD47 and CD19 antibody) , TG Therapeutics, Inc. , TG-1801 OR TG-1801 + ublituximab , B-cell lymphoma , 16 , December 2022 , NCT03804996 , I , Recruiting.

Techniques: Recombinant, Transformation Assay, Mutagenesis

Journal: Nature Communications

Article Title: Cancer cell-expressed SLAMF7 is not required for CD47-mediated phagocytosis

doi: 10.1038/s41467-018-08013-z

Figure Lengend Snippet: Tumor-expressing SLAMF7 is not required for induction of phagocytosis upon CD47-targeting treatment in DLBCL cells. a Percentage of phagocytosis of DLBCL cell lines by allogeneic human macrophages primed with LPS/IFN-γ upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 3–5). b Representative microscopy pictures of phagocytosis of tumor cells by macrophages primed with LPS/IFN-γ upon 3 h treatment with CD47 F(ab′)2 (left, MØ + V450-labeled OCIly3 cells, right, MØ + pHrodogreen-labeled SUDHL5 cells). Scale bar = 20 µm. c Representative graphs of flow cytometric analysis for phagocytosis of tumor cells by macrophages with LPS/IFN-γ upon 3 h treatment with CD47 F(ab′)2 (left, MØ + SUDHL5, right, MØ + SUDHL10). d Quantification of phagocytosis of DLBCL cell lines by flow cytometric analysis. Experimental setting is the same as in ( a ) ( n = 3–4). e Percentage of different types of macrophages from cibersort fraction of DLBCL biopsies ( n = 1804). f Percentage of phagocytosis of DLBCL cell lines by allogeneic type 0 human macrophages upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 4–6). g Percentage of phagocytosis of DLBCL cell lines by allogeneic human macrophages primed with IL-10 upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 4–6). Statistics was performed using paired Student’s t -test. n.s. = not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Error bars stand for standard deviation (SD)

Article Snippet: APC-labeled anti-CD3, FITC-labeled anti-CD19 and APC-labeled anti-CD47 were purchased from Immunotools (Germany).

Techniques: Expressing, Microscopy, Labeling, Standard Deviation

Journal: Nature Communications

Article Title: Cancer cell-expressed SLAMF7 is not required for CD47-mediated phagocytosis

doi: 10.1038/s41467-018-08013-z

Figure Lengend Snippet: Efficacy of CD47-targeting antibodies in B-cell malignant cells does not correlate with SLAMF7 expression. a Experimental phagocytosis of DLBCL lines by macrophages either upon CD47 F(ab′)2 treatment or inhibrix treatment ( n = 3). Box plot contains center line representing median and whiskers representing 5–95%. b Representative microscopy pictures of phagocytosis of primary DLBCL cells by autologous macrophages upon 3 h treatment with inhibrix. Quantification of phagocytosis of primary DLBCL and MCL cells by autologous macrophages. c Quantification of surface SLAMF7 expression on five NHL lines ( n = 3). d Percentage of phagocytosis of NHL cell lines by allogeneic human macrophages primed with LPS/IFN-γ upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 3–4). e Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by CD47 F(ab′)2 in NHL and DLBCL cell panel ( n = 3). f Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by anti-CD47 antibody inhibrix in DLBCL cell panel ( n = 3–4). g Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by Rituximab in NHL and DLBCL cell panel ( n = 3). h Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by the combinatory treatment of Rituximab and Inhibrix in DLBCL cell panel ( n = 3). i Experimental phagocytosis of tumor cells by macrophages upon RTX treatment or combination treatment with inhibrix ( n = 3). Experiments with primary patient-derived samples were performed in triplicates. Statistics was performed using paired Student’s t -test. n.s. = not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Error bars stand for standard deviation (SD)

Article Snippet: APC-labeled anti-CD3, FITC-labeled anti-CD19 and APC-labeled anti-CD47 were purchased from Immunotools (Germany).

Techniques: Expressing, Microscopy, Derivative Assay, Standard Deviation

Journal: Nature Communications

Article Title: Cancer cell-expressed SLAMF7 is not required for CD47-mediated phagocytosis

doi: 10.1038/s41467-018-08013-z

Figure Lengend Snippet: mRNA expression of SLAMF7 does not, but of CD47 does, associate with survival in DLBCL patients. a Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low SLAMF7-expressing quartiles. b Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low CD47-expressing quartiles. c Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low SLAMF7-expressing quartiles additionally sorted on high and low expression of CD47. d Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low CD47-expressing quartiles additionally sorted on high and low expression of SLAMF7

Article Snippet: APC-labeled anti-CD3, FITC-labeled anti-CD19 and APC-labeled anti-CD47 were purchased from Immunotools (Germany).

Techniques: Expressing

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