anti fitc-labeled cd47  (Elabscience Biotechnology)

Journal: bioRxiv

Article Title: Immune-Derived THBS1-CD47 Axis Induces Cellular Senescence and Suppresses Osteogenesis in Diabetic Periosteum

doi: 10.1101/2025.11.19.689158

Figure Lengend Snippet: ( A ) CellChat comparison analysis illustrating changes in the number of immune cells regulating osteolineage cells in the periosteum of diabetic and control group. Red lines indicate increased regulation, while blue lines indicate decreased regulation, with line thickness representing the interaction strength. ( B ) Differential ligand-receptor pairs between T2DM and CTRL periosteal cells. ( C ) Violin plots of periosteal expression levels of ligands and receptors involved in THBS1 and OSM signaling. Statistical significance was assessed using MAST. ( D ) Flow cytometry analysis measuring CD47 protein expression in periosteal cells from CTRL (n=3 mice) and 3-month T2DM (n=3 mice). ( E ) Bar graph comparing the relative abundance of CD47 hi and CD47 low periosteal cells in 3-month T2DM and CTRL mice. ( F ) ELISA quantification of THBS1 levels in serum from T2DM (n=8 mice) and CTRL (n=8 mice). ( G ) Immunofluorescence staining of THBS1 in periosteum, with quantification of fluorescence intensity. CTRL, n = 3 mice; T2DM, n = 4 mice. Scale bar: 100 μm. ( H ) Relative proportion of bone marrow T2DM and CTRL in each cell cluster. ( I - K ) Expression levels of Thbs1 and Cd47 in immune cells and osteolineage cells of bone marrow. Statistical significance was assessed using MAST. ( I ) Differential ligand-receptor interactions between immune cells and osteo-CAR cells in the bone marrow of T2DM and CTRL group. Bar graphs represent mean ± S.D. Statistical analysis was performed using unpaired Student’s t-test unless otherwise stated.

Article Snippet: Thrombospondin-1 blocking antibody (15 μl of a 250 μg/ml solution diluted in sterile PBS, Thermo Fisher, clone A6.1) or CD47 blocking antibody (15μg, BioXcell, BE0283) was injected into the fracture site of diabetic C57BL/6J mice.

Techniques: Comparison, Control, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Immune-Derived THBS1-CD47 Axis Induces Cellular Senescence and Suppresses Osteogenesis in Diabetic Periosteum

doi: 10.1101/2025.11.19.689158

Figure Lengend Snippet: ( A ) Representative 3D micro-CT reconstructions of femoral fracture callus formation at day 14 post-fracture in CTRL+IgG (n = 3), T2DM+IgG (n = 3), and T2DM + a-CD47 (n = 4) groups. Scale bar: 1 mm. ( B ) Quantitative micro-CT analysis of bone mineral density (BMD), trabecular number (Tb.N), bone volume fraction (BV/TV), and trabecular separation (Tb.Sp). ( C ) Representative histological staining of fracture callus with Masson’s trichrome and Safranin O/Fast Green, showing improved callus formation in T2DM+a-CD47 compared to T2DM+IgG. Higher magnification views of the areas outlined with dashed boxes. Thick and thin scale bar: 500 μm. Data are presented as mean ± S.D. At least three biological replicates were analyzed. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. *p<0.05; **p<0.01, ***p<0.001

Article Snippet: Thrombospondin-1 blocking antibody (15 μl of a 250 μg/ml solution diluted in sterile PBS, Thermo Fisher, clone A6.1) or CD47 blocking antibody (15μg, BioXcell, BE0283) was injected into the fracture site of diabetic C57BL/6J mice.

Techniques: Micro-CT, Staining

Journal: Cell Reports Medicine

Article Title: Inhibiting KRAS with CD47 and immune checkpoint overcomes intrinsic resistance to combined KRAS and immune checkpoint inhibitor therapy

doi: 10.1016/j.xcrm.2025.102317

Figure Lengend Snippet: Inhibition of KRAS upregulates the expression of CD24 and CD47 (A) NCI-H358 and LU65 KRAS G12C-mutant cancer cell lines were treated with DMSO or 1 μM sotorasib at the indicated time points. Cell lysates were probed with the indicated antibodies. Western blot was repeated three independent times with similar results. (B and C) The mean fluorescent intensity (MFI) of CD47 (B) and CD24 (C) on NCI-H358, LU65, and GP5d cells treated with DMSO or 1 μM sotorasib for 72 h. The data represent MFI ± SD of 3 wells, ∗ p < 0.05 by t test. (D) Flow cytometric phagocytosis assay of THP-1-derived macrophages against NCI-H358 and LU65 cells and human monocyte-derived macrophages against NCI-H358 cells. Tumor cells were treated with DMSO or 1 μM sotorasib for 72 h and then stained with CFSE. THP-1-derived macrophages and human monocyte-derived macrophages were labeled with PKH-26 staining solution. Subsequently, tumor cells were co-cultured with macrophages at a 1:1 ratio for 2 h. The percentage of both CFSE- and PKH-positive cells among PKH-positive cells was measured using flow cytometry and compared between DMSO- and sotorasib-treated cells. The data represent mean ± SD of 3 independent experiments, ∗ p < 0.05 by t test. (E) The MFI of CD47 on LLC Nras KO cells following inhibition of mutant KRAS. For in vitro analysis, LLC Nras KO cells were treated with DMSO or 1 μM sotorasib for 72 h. For in vivo experiments, enhanced green fluorescent protein (EGFP)-labeled LLC Nras KO syngeneic lung tumors were treated with vehicle or 100 mg/kg sotorasib for 3 days. Among the EGFP-positive cells, the MFI of CD47 was measured using flow cytometry with anti-CD47 antibodies. The data represent MFI ± SD of 3 independent wells or mice, respectively, ∗ p < 0.05 by t test. (F) Flow cytometric phagocytosis assay of murine peritoneal macrophages against LLC Nras KO cells assessed as in (E). The data represent mean ± SD of 3 independent experiments, ∗ p < 0.05 by t test. (G) EGFP-labeled LLC Nras KO syngeneic lung tumors were treated with vehicle or 100 mg/kg sotorasib for 3 days. Then tumors were collected, dissociated, and labeled with F4/80 antibody. The percentage of both EGFP- and F4/80-positive cells among F4/80-positive cells was measured using flow cytometry and compared between vehicle- and sotorasib-treated mice. The data represent mean ± SD of 3 independent mice, ∗ p < 0.05 by t test.

Article Snippet: Sotorasib, adagrasib and MRTX-1133 were obtained from Chemgood, and the MIAP410 monoclonal InVivoMAb anti-mouse/human/rat CD47 (IAP) was purchased from BioXCell.

Techniques: Inhibition, Expressing, Mutagenesis, Western Blot, Phagocytosis Assay, Derivative Assay, Staining, Labeling, Cell Culture, Flow Cytometry, In Vitro, In Vivo

Journal: Cell Reports Medicine

Article Title: Inhibiting KRAS with CD47 and immune checkpoint overcomes intrinsic resistance to combined KRAS and immune checkpoint inhibitor therapy

doi: 10.1016/j.xcrm.2025.102317

Figure Lengend Snippet: FOXA1 binds to the super-enhancer region of CD47 and regulates its expression (A) ATAC-seq signal for the CD47 gene in NCI-H358 cells. Arrows are pile-up reads in super-enhancer regions (peak A, blue; and peak B, green) and a promoter region (peak C, orange). (B) Flowchart identifying transcription factors that regulate CD47 is shown on the left. Fold change in expression of each candidate transcription factor in NCI-H358 cells transfected with constitutively active YAP (YAP S6A) or in NCI-H358 cells treated with 1 μM sotorasib for 72 h relative to NCI-H358 cells is shown on the right. (C) qPCR analysis of mRNA levels of FOXA1 normalized to ubiquitin following treatment with 1 μM sotorasib for 72 h in NCI-H358 and LU65 cells. Data are mean ± SD ( n = 3 independently treated cell cultures, two-sided Student’s t test, ∗ p < 0.05). (D) NCI-H358 and LU65 cells were treated with DMSO or 1 μM sotorasib at the indicated time points. Cell lysates were probed with the indicated antibodies. (E and F) NCI-H358 and LU65 cells transduced with control sgRNA or sgFOXA1 were treated with 1 μM sotorasib for 72 h. Cell lysates were probed with the indicated antibodies. Western blot was repeated 2 independent times with similar results (E). The MFI of CD47 on NCI-H358 and LU65 cells is shown (F). The data represent MFI ± SD of 3 wells, ∗ p < 0.05 by t test. (G) ChIP-qPCR analysis shows significantly more enrichment of FOXA1 at the super-enhancer regions of the CD47 gene. Data are mean ± SD ( n = 3 independently treated cell cultures, two-sided Student’s t test, ∗ p < 0.05).

Article Snippet: Sotorasib, adagrasib and MRTX-1133 were obtained from Chemgood, and the MIAP410 monoclonal InVivoMAb anti-mouse/human/rat CD47 (IAP) was purchased from BioXCell.

Techniques: Expressing, Transfection, Ubiquitin Proteomics, Transduction, Control, Western Blot, ChIP-qPCR

Journal: Cell Reports Medicine

Article Title: Inhibiting KRAS with CD47 and immune checkpoint overcomes intrinsic resistance to combined KRAS and immune checkpoint inhibitor therapy

doi: 10.1016/j.xcrm.2025.102317

Figure Lengend Snippet: Inhibition of KRAS and CD47 induces a phenotypic change in macrophages (A–C) Flow cytometric phagocytosis assay of THP-1-derived macrophages against NCI-H358 and LU65 cells (A), human monocyte-derived macrophages against NCI-H358 cells (B), and murine peritoneal macrophages against LLC Nras KO cells (C). Tumor cells were treated with DMSO or 1 μM sotorasib for 72 h and then co-cultured with macrophages at a 1:1 ratio for 2 h with or without 2 μg/mL anti-CD47. The data represent mean ± SD of 3 independent experiments, ∗ two-sided Student’s t test with Bonferroni correction, p < 0.05. (D) Phagocytosis assay in vivo . EGFP-labeled LLC Nras KO syngeneic lung tumors were treated with vehicle, sotorasib (100 mg/kg), anti-CD47 (100 μg/body), or the combination of these drugs at the same doses for 3 days. The data represent mean ± SD of 3 independent mice, ∗ two-sided Student’s t test with Bonferroni correction, p < 0.05. (E) LLC Nras KO syngeneic lung tumors were treated with vehicle, sotorasib (100 mg/kg), anti-CD47 (100 μg/body), or the combination of these drugs at the same doses for 3 days. Tumors were harvested and subjected to fluorescence immunostaining with the indicated antibodies. Scale bars, 100 μm. (F) LLC Nras KO syngeneic lung tumors were treated as in (E). Gene expression of Adgre1 was visualized as yellow. Scale bars, 1 mm. (G) UMAP visualization of merged sequencing profiles from tumors of each cohort with cells colored and labeled according to cell type. (H) A 100% stacked bar graph showing the proportion of macrophages classified into each cluster. (I) The spatial distribution of each macrophage cluster in each cohort. Scale bars, 1 mm. (J) The top 15 most significantly upregulated genes in clusters 1, 3, and 11. The complete list is included in . (K) LLC Nras KO syngeneic lung tumors were treated as in (E) and subjected to fluorescence immunostaining with the indicated antibodies. Scale bars, 100 μm. (L) Phagocytosis assay in vivo . Percentage of phagocytic macrophages was defined by the ratio of EGFP-positive cells among iNOS- or CD206-positive macrophages, respectively, in EGFP + LLC Nras KO syngeneic lung tumors. The data represent mean ± SD of 3 mice, ∗ p < 0.05 by t test.

Article Snippet: Sotorasib, adagrasib and MRTX-1133 were obtained from Chemgood, and the MIAP410 monoclonal InVivoMAb anti-mouse/human/rat CD47 (IAP) was purchased from BioXCell.

Techniques: Inhibition, Phagocytosis Assay, Derivative Assay, Cell Culture, In Vivo, Labeling, Fluorescence, Immunostaining, Gene Expression, Sequencing

Journal: Cell Reports Medicine

Article Title: Inhibiting KRAS with CD47 and immune checkpoint overcomes intrinsic resistance to combined KRAS and immune checkpoint inhibitor therapy

doi: 10.1016/j.xcrm.2025.102317

Figure Lengend Snippet: Phagocytosis induces PD-L1 expression on macrophages (A) qPCR analysis of mRNA levels of PD-L1 normalized to ubiquitin in non-phagocytic and phagocytic macrophages co-cultured with NCI-H358 cells for 24 h. Data are mean ± SD ( n = 3 independently treated cell cultures, two-sided Student’s t test, ∗ p < 0.05). (B) The upregulation of PD-L1 MFI following phagocytosis of THP-1-derived macrophages by co-culture with NCI-H358 and LU65 cells for 2 h. The data represent MFI ± SD of 3 wells, two-sided Student’s t test, ∗ p < 0.05. (C) Human monocyte-derived macrophages were co-cultured with NCI-H358 cells for 2 h, and PD-L1 expression was compared between non-phagocytic and phagocytic macrophages. The data represent MFI ±SD of 3 independently treated cell cultures, two-sided Student’s t test, ∗ p < 0.05. (D) The upregulation of PD-L1 MFI following phagocytosis of murine peritoneal macrophages by co-culture with LLC Nras KO cells for 2 h. The data represent MFI ± SD of 3 wells, two-sided Student’s t test, ∗ p < 0.05. (E) Upregulation of PD-L1 MFI on phagocytic macrophages in vivo . LLC Nras KO syngeneic lung tumors were treated with vehicle, sotorasib (100mg/kg), anti-CD47 (100μg/body), or the combination of these drugs at the same doses for 3 days. The data represent MFI ± SD of 3 mice, two-sided Student’s t test with Bonferroni correction, ∗ p < 0.05. (F) PD-L1 expression in the tumor microenvironment of LLC Nras KO syngeneic lung tumors. Tumors were harvested and subjected to fluorescence immunostaining with the indicated antibodies. Scale bars, 50 μm. (G) LLC Nras KO syngeneic lung tumors were treated with vehicle, sotorasib (100 mg/kg), anti-CD47 (100 μg/body), or the combination of these drugs at the same doses for 3 days. Tumors were harvested and subjected to fluorescence immunostaining with the indicated antibodies. Scale bars, 100 μm. (H) Quantitative analyses of PD-L1-positive cells. The y axis represents the number of positive cells for each determinant per ×20 microscopic field ( n = 5 mice/group, with at least 3 fields per slide). The data are presented as the mean ± SD. ∗ p < 0.05 by Mann-Whitney U tests with Bonferroni correction.

Article Snippet: Sotorasib, adagrasib and MRTX-1133 were obtained from Chemgood, and the MIAP410 monoclonal InVivoMAb anti-mouse/human/rat CD47 (IAP) was purchased from BioXCell.

Techniques: Expressing, Ubiquitin Proteomics, Cell Culture, Derivative Assay, Co-Culture Assay, In Vivo, Fluorescence, Immunostaining, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Inhibiting KRAS with CD47 and immune checkpoint overcomes intrinsic resistance to combined KRAS and immune checkpoint inhibitor therapy

doi: 10.1016/j.xcrm.2025.102317

Figure Lengend Snippet: KRAS inhibition combined with anti-CD47 and immune checkpoint blockade results in enhanced efficacy (A) Tumor-associated macrophages suppressed T cell activation that was relieved by anti-PD-L1 antibody. CD8 T cells isolated from C57BL/6 mouse spleen were seeded into plates pre-coated with anti-CD3 antibody and cultured for 24 h in medium supplemented with the anti-CD28 antibody. Tumor-associated macrophages were generated from co-culture of mouse peritoneal macrophages with LLC Nras KO cells for 24 h. Then, tumor-associated macrophages, CD8 T cells, and a mixture of these cells were cultured with or without 10 μg/mL anti-PD-L1 antibody for 24 h. T cell activation was measured by IFN-γ secretion in each media. The data represent mean ± SD of 3 co-cultures, two-sided Student’s t test with Bonferroni correction, ∗ p < 0.05. (B–F) Representative immunofluorescence images (B) and quantitative analyses (C–F) showing indicated immune cells infiltrating tumors. The y axis represents the number of positive cells for each determinant per ×20 microscopic field ( n = 5 mice/group, with at least 3 fields per slide). LLC Nras KO syngeneic lung tumors were treated with vehicle, sotorasib (100 mg/kg), anti-CD47 antibody (100 μg/body), atezolizumab (20 mg/kg), or the combination of these drugs as indicated for 3 days. Tumors were harvested and subjected to fluorescence immunostaining with the indicated antibodies. Scale bars: 20 μm. The data are presented as the mean ± SD. ∗ p < 0.05 by Mann-Whitney U tests with Bonferroni correction. (G) LLC Nras KO tumors were treated with vehicle, 50 mg/kg sotorasib daily, anti-CD47 antibody 50 μg/body three times a week, atezolizumab 10 mg/kg twice a week, or a combination of these drugs as indicated. Treatments were finished on day 56, and mice were followed until 1 year. Kaplan-Meier survival curve of mice in each group. Significance was determined by a log rank test, ∗ p < 0.001.

Article Snippet: Sotorasib, adagrasib and MRTX-1133 were obtained from Chemgood, and the MIAP410 monoclonal InVivoMAb anti-mouse/human/rat CD47 (IAP) was purchased from BioXCell.

Techniques: Inhibition, Activation Assay, Isolation, Cell Culture, Generated, Co-Culture Assay, Immunofluorescence, Fluorescence, Immunostaining, MANN-WHITNEY