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: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: CD47 (cluster of differentiation) mediates TSP1 (thrombospondin-1)-induced inhibition of lymphangiogenesis. A , Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the relative mRNA levels of CD47 and CD36 in human lymphatic endothelial cells (HLECs) and human aortic endothelial cells (HAoEC). Bar graph represents mRNA levels in comparison to CD36 (n=9). B , Control and CD47 siRNA-treated lymphatic endothelial cells (LECs; 48 h) were utilized to quantify CD47 transcript expression using qRT-PCR (n=6). C , WST (water-soluble tetrazolium)-1 assay was conducted to investigate the effects of CD47 silencing on LEC proliferation in response to TSP1 treatment as described in Figure 2A. Data are representative of 3 independent experiments performed at least in quadruplicate. D , Control and CD47 -silenced LECs were used to evaluate cell migration. Scale bar, 200 µm. xBar graph represents the percentage of migrated cells (n=3–4). E through G , Control and CD47 -silenced cells were pretreated as in Figure 2D and seeded in wells of a Matrigel-coated plate in basal medium containing VEGF (vascular endothelial growth factor)-C±TSP1 and tube formation determined. Representative images of tube formation are shown ( E ). Scale bar, 1000 µm. Tube length ( F ) and number of branching points ( G ) quantified (n=7). H , Wild-type male mice were injected SC with Matrigel solutions premixed with either VEGF-C, VEGF-C+TSP1+IgG, or VEGF-C+TSP1+CD47-blocking antibody. Plugs were isolated after 10 days, sectioned, and immunostained for LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1). Representative images of LYVE-1 staining of the cross-sections of the Matrigel plugs and quantification of LYVE-1–positive area are shown (n=5–7). Scale bar, 20 μm. Statistical analyses were performed using 2-way ANOVA ( A , C , D , F , and G ) with Bonferroni ( A ), Tukey ( C , F , and G ), and Sidak ( D ) multiple comparisons test, 2-tailed unpaired Student t test ( B ), and Kruskal-Wallis test for multiple comparisons ( H ). Data represent mean±SEM.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Control, Expressing, Migration, Injection, Blocking Assay, Isolation, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: TSP1 (thrombospondin-1)-induced CD47 (cluster of differentiation) activation blocks VEGF (vascular endothelial growth factor)-C–stimulated lymphangiogenic signaling. A through D , Lymphatic endothelial cells (LECs) were pretreated with TSP1 (22 nM, 16 h) in 0.5% fetal bovine serum (FBS) containing basal media MV2, stimulated with VEGF-C (15 min), and cell lysates subjected to Western blot analysis. A , Representative Western blot images are shown. B through D , Bar diagrams represent mean protein levels expressed as a ratio of phospho-to-total proteins, AKT ( B ), eNOS ( C ), and ERK1/2 ( D ; n=3). E , LECs were treated with control or CD47 -siRNA (48 h) and immunoblotting done to determine CD47 expression (ab175388). F through I , Control or CD47 -siRNA–treated cells were treated as in Figure 4A and Western blot experiments executed. F , Representative Western blot images are shown. G through I , Bar diagrams represent pAKT/total AKT ( G ), peNOS/total eNOS ( H ), and pERK1/2/total ERK1/2 ( I ; n=3). J , Control or CD47 -silenced LECs were pretreated with TSP1 (22 nM, 4 h), stimulated with VEGF-C (100 ng/mL, 1 h) and analyzed for NO (nitric oxide) production using DAF-FM diacetate. K , Control or CD47 -silenced LECs were pretreated with vehicle or TSP1 (1 h), incubated with H2DCFDA solution, and fluorescence analyzed using flow cytometry. Statistical analyses were performed using 1-way ANOVA ( B–D ), 2-way ANOVA with Tukey test for multiple comparisons ( G–I and K ), and 2-tailed unpaired Student t test ( J ). Data represent mean±SEM.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Activation Assay, Western Blot, Control, Expressing, Incubation, Fluorescence, Flow Cytometry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: Lymphatic endothelial cell (LEC)–specific Cd47 deficiency reduces atherosclerotic lesion formation. A through M , Male Cd47 WT and Cd47 ΔLEC mice were injected with AAV (adeno-associated virus) 8- PCSK9 IP, fed a Western diet for 16 weeks and atherosclerosis analyzed. A , Representative in situ images of aortic arch (yellow arrowheads point to atherosclerotic lesions). Scale bar, 2 mm. B , Representative en face oil red O (ORO) staining of aorta. Scale bar, 5 mm. C , Quantification of plaque area in aorta (n=13–14). D through G , Bar diagrams show plasma total cholesterol ( D ), plasma triglycerides ( E ), fasting blood glucose levels ( F ), and body composition (fat, lean, and fluid mass; G ; n=8–13). H , Representative images of aortic root cross-sections stained with H&E (neointima area), ORO (lipid accumulation), Masson trichrome (collagen content), CD68 (cluster of differentiation; macrophage burden), and necrotic area (encircled in red). Scale bar, 200 µm. I through M , Bar diagrams show neointima area ( I ), lipid deposition ( J ), collagen content ( K ), macrophage accumulation ( L ), and necrotic area ( M ) in aortic root sections (n=5–9). Statistical analyses were performed using a 2-tailed unpaired Mann-Whitney U test ( C , E , and M ), 2-tailed unpaired Student t test ( D , F , and I–L ), and 2-way ANOVA followed by Sidak post hoc test ( G ). Data represent mean±SEM.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Injection, Virus, Western Blot, In Situ, Staining, Clinical Proteomics, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: Lymphatic endothelial cell (LEC)–specific deletion of Cd47 (cluster of differentiation) in mice increases arterial lymphatic vessel (LV) density. A through D , Aortic root cross-sections from male AAV (adeno-associated virus) 8- PCSK9 –injected Cd47 WT and Cd47 ΔLEC mice (16-wk Western diet) were immunostained for CD68, iNOS (inducible NO [nitric oxide] synthase), Arg1 (arginase 1), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1). Nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole; blue). Representative confocal images of iNOS (green; A ) and CD68 (red); Arg1 (green; B ) and CD68 (red; scale bar, 50 µm); and LYVE-1 staining (red; C ; scale bar, 20 µm) are shown (n=5–6). Statistical analyses were performed using a 2-tailed unpaired Student t test ( A and B ) and a Mann-Whitney U test ( C ). Data represent mean±SEM. A indicates adventitia; M, media; and P, plaque.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Virus, Injection, Western Blot, Staining, MANN-WHITNEY

Journal: Journal of leukocyte biology

Article Title: Contact-dependent, polarized acidification response during neutrophil–epithelial interactions

doi: 10.1002/JLB.3MA0422-742R

Figure Lengend Snippet: Role of PMN TEM in extracellular acidification. (A) Quantification of MPO activity at T90 ( n = 4). (B) Changes in extracellular pH. 1.5 × 10 6 PMNs were transmigrated basolateral to apical across confluent T84 monolayers in the presence or absence of 10 μ g/ml anti-CD47, anti-CD11b, anti-CD55, or IgG controls and the extracellular pH was recorded every minute for 90 min. (C) At T90, the difference between starting and ending pH were calculated. Data are expressed as change in pH. Rat and mouse IgG controls were pool in the IgG+fMLP group. Data are expressed as pH ( n = 4). (D) Changes in extracellular pH. 1.5 × 10 6 PMNs were transmigrated apical to basolateral across confluent T84 monolayers plated as inserts in the presence or absence of 10 μ g/ml anti-CD47, anti-CD11b, anti-CD55, or IgG controls and the extracellular pH was recorded every minute for 90 min. n = number of independent experiments performed, separate passages of cells were used for each experiment. The data for each experiment were pooled and expressed as the mean ± SEM and p value determined by ANOVA. *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-CD47 (B6H12) and IgG1 mouse isotype control monoclonal antibody (10 μ g/ml) was obtained from Fisher Scientific (Hampton, NH).

Techniques: Activity Assay