Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 1. CD47 levels in human lung adenocarcinoma cells were increased following tumor metastasis (A and B) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the lymph nodes. (C and D) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the liver. (A) and (C) showed representative IHC images for CD47 staining from 14 to 5 lung adenocarcinoma tissue samples, respectively. Data were presented as means ± SDs. **p < 0.01. Scale bar, 50 mm.

Article Snippet: Then, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD47 antibody (sc-21786, Santa Cruz Biotechnology, Dallas, TX, USA) on ice 284 Molecular Therapy: Oncolytics Vol.

Techniques: Comparison, Expressing, Staining

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 2. Induction of CD47 by IFN-g in human lung cancer cells (A) Flow cytometry analysis of CD47 surface expression in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Right: Representative image; left: quantitative analysis. (B) Immunofluorescence analysis of CD47 expression in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Scale bar, 20 mm. (C) Western blot analysis of CD47 expression in human lung cancer cell lines after IFN-g treatment. (D) qRT-PCR analysis of CD47 mRNA in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Left: Representative image; right: quantitative analysis. Data from more than 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Then, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD47 antibody (sc-21786, Santa Cruz Biotechnology, Dallas, TX, USA) on ice 284 Molecular Therapy: Oncolytics Vol.

Techniques: Flow Cytometry, Expressing, Incubation, Recombinant, Western Blot, Quantitative RT-PCR

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 3. Identification of genes involved in the IFN signaling pathway that upregulates CD47 expression (A) qRT-PCR analysis of various genes involved in the IFN signaling pathway in A549 cells upon incubation with re- combinant IFN-g (100 ng/mL, 24 h). (B) Normalized luciferase reporter expression of A549 cells transduced with different sets of lentiviral shRNA constructs. Upper: Schematic representation of the CD47-Prom-Firefly Lucif- erase-EF1AProm-Renilla luciferase constructs used to generate the reporter cell lines. Lower: Quantitative anal- ysis. (C) IRF1 protein levels in human lung cancer cells upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Upper: Representative image; lower: quantitative anal- ysis. Data from more than 3 independent experiments were presented as means ± SDs. ***p < 0.001.

Article Snippet: Then, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD47 antibody (sc-21786, Santa Cruz Biotechnology, Dallas, TX, USA) on ice 284 Molecular Therapy: Oncolytics Vol.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Luciferase, Transduction, shRNA, Construct, Recombinant

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 4. IFN-g induces CD47 expression in lung cancer cells through IRF-1 (A) Sequence of the CD47 promoter showing the position of the putative IRF-1-binding site. (B) Reporter assay for putative IRF-1 binding. The results were normalized to relative luciferase units (RLUs). (C) ChIP assay analyzing the CD47 promoter in A549 cells. The results were normalized to the input. (D and E) IRF-1 mRNA (D) and protein (E) levels in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-g treatment. (F and G) CD47 level in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-g treatment detected by flow cytometry (F) and immunofluorescence (G). Scale bar, 10 mm. (H) Western blot analysis of CD47 levels in A549 cells transfected with IRF-1-specific or scramble shRNA. Left: Representative image; right: quantitative analysis. Data from 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Then, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD47 antibody (sc-21786, Santa Cruz Biotechnology, Dallas, TX, USA) on ice 284 Molecular Therapy: Oncolytics Vol.

Techniques: Expressing, Sequencing, Binding Assay, Reporter Assay, Luciferase, Transfection, shRNA, Cytometry, Western Blot

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 5. IFN-g promotes lung cancer cell metastasis by upregulating CD47 expression in vitro (A) Scratch-wound healing assay in A549 cells (A549-WT) and CD47-knockout A549 cells (A549-CD47-KO) were treated with/without IFN-g. (B) Transwell assay in A549 cells (A549-WT) and A549-CD47-KO cells were treated with/without IFN-g. Data from 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 200 mm.

Article Snippet: Then, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD47 antibody (sc-21786, Santa Cruz Biotechnology, Dallas, TX, USA) on ice 284 Molecular Therapy: Oncolytics Vol.

Techniques: Expressing, In Vitro, Wound Healing Assay, Knock-Out, Transwell Assay

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 6. IFN-g promotes human lung cancer cell metastasis in immunodeficient mice by upregulating CD47 (A) A549 cells (A549-WT) and A549-CD47-KO were engrafted in the lungs of the BALB/c-nude mice. Three weeks post-engraftment, the mice were randomly divided into 2 groups. One group was administered with IFN-g (10 ng/mouse, injected once every 2 days) (A549-WT + IFN-g; A549-CD47-KO + IFN-g), and the other group without IFN-g injection was served as control (A549-WT; A549-CD47-KO). After 5 weeks, the mice were sacrificed to analyze tumor growth and metastasis. (B and C) H&E staining and human CD47 immune staining in mouse lungs (B) and livers (C). In both (B) and (C), left: representative images; right: quantification of tumor nodules. Data were presented as means ± SDs. *p < 0.05, ***p < 0.001. NS, no significance.

Article Snippet: Then, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD47 antibody (sc-21786, Santa Cruz Biotechnology, Dallas, TX, USA) on ice 284 Molecular Therapy: Oncolytics Vol.

Techniques: Injection, Control, Staining

Journal: Advanced Science

Article Title: Protecting Against Postsurgery Oral Cancer Recurrence with an Implantable Hydrogel Vaccine for In Situ Photoimmunotherapy

doi: 10.1002/advs.202309053

Figure Lengend Snippet: Phototherapeutic effects and light‐controlled drug release of APHP‐CCCA. A) Time‐dependent inhibition of Cal33 cell viability by photothermal therapy using 100 µg mL −1 APHP‐CCCA upon 808 nm laser irradiation at 1.25 W cm −2 for different durations (5, 10, 15, and 20 min; NIR‐5, NIR‐10, NIR‐15, and NIR‐20). B) Quantitative analysis of intracellular PpIX concentration in Cal33 cells treated with 100 µg mL −1 APHP‐CCCA after different durations of 808 nm laser irradiation. C) Dose‐response curve from SPR measurements showing binding of aCD47 released from APHP‐CCCA without laser irradiation to immobilized CD47 protein. D) Dose‐response curve from SPR measurements showing binding of aCD47 released from APHP‐CCCA upon 808 nm laser irradiation at 1.25 W cm −2 for 5 min to immobilized CD47 protein. E) SPR sensorgrams showing binding of free aCD47, aCD47 released from aCD47@CaCO 3 nanoparticles or APHP‐CCCA hydrogel after 808 nm laser irradiation for 5 min to immobilized CD47 protein. F) Dissociation equilibrium constant (K D ) values of aCD47 released from APHP‐CCCA upon 808 nm laser irradiation at 1.25 W cm −2 for different durations determined by SPR analysis. G) Quantitative analysis of intracellular PpIX concentration in Cal33 cells treated with APHP or APHP‐CCCA and irradiated with 808 nm laser at 1.25 W cm −2 for 5 min and/or 660 nm laser at 40 mW cm −2 for 20 min followed by 24 h culture. H) Viability of Cal33 cells treated with APHP or APHP‐CCCA and irradiated with 808 nm laser at 1.25 W cm −2 for 5 min and/or 660 nm laser at 40 mW cm −2 for 20 min followed by 24 h culture. I) ROS levels in Cal33 and HN6 cells determined by flow cytometry after treatment with 660 nm laser at 40 mW cm −2 for different durations (0, 2.5, 5, 7.5, and 10 min). J) Viability of Cal33 cells treated with APHP‐CCCA and irradiated with 808 nm laser at 1.25 W cm −2 for 5 min, followed by irradiation with 660 nm laser at 40 mW cm −2 for different durations (0, 2.5, 5, 7.5, and 10 min). K) Representative TEM images of the ultrastructure of Cal33 cells treated with APHP‐CCCA with no NIR irradiation, 5 min of 808 nm laser exposure, or 5 min of 808 nm plus 5 min of 660 nm laser irradiation. Scale bar, 2 µm, 500 nm, and 100 nm. L) Intratumoral pH values on each day for 5 d following surgery. M) Representative fluorescence images of intracellular PpIX in residual tumors on each day for 5 d following surgery after 5 min of 808 nm plus 5 min of 660 nm laser irradiation. Red, PpIX; blue, DAPI. Scale bar, 20 µm. N) Representative fluorescence images of CD47 expression in residual tumor tissue on each day for 5 d following surgery after 5 min of 808 nm plus 5 min of 660 nm laser irradiation. Yellow, CD47; blue, DAPI. Scale bar, 20 µm. Data are presented as the mean ± SD; n = 3 independent experiments. p values were determined by two‐way ANOVA, Tukey's multiple‐comparison test (ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Recombinant human CD47 protein was obtained from MedChemExpress (MCE, USA).

Techniques: Inhibition, Irradiation, Concentration Assay, Binding Assay, Flow Cytometry, Fluorescence, Expressing, Comparison

Journal: Advanced Science

Article Title: Protecting Against Postsurgery Oral Cancer Recurrence with an Implantable Hydrogel Vaccine for In Situ Photoimmunotherapy

doi: 10.1002/advs.202309053

Figure Lengend Snippet: In vivo tumor recurrence suppression efficacy of APHP‐CCCA hydrogel implantation postsurgery.A) Schematic illustration of the animal experimental design. NIR irradiation: 808 nm laser at 1.25 W cm −2 for 5 min and/or 660 nm laser at 40 mW cm −2 for 5 min. B) Individual tumor growth curves of mice in different groups over 30 d following surgery. RR: recurrence rate. C) Average tumor growth curves of different groups during 30 d after surgery. Data are shown as mean ± SEM ( n = 5 mice per group). D) Percentage of mice with tumor recurrence in each group at 30 d after surgery ( n = 5 mice per group). E) Tumor weights of recurrent tumors on day 30 after surgery. Data are shown as mean ± SD ( n = 5 mice per group). F) Long‐term survival percentages of mice in each group over 60 d following surgery ( n = 5 mice per group). G) H&E staining of residual tumors in each group at 30 d after surgery; scale bar, 200 µm. Ki67 immunohistochemical staining of residual tumors in each group at third day after surgery; scale bar, 200 µm. Representative fluorescence images showing intracellular PpIX, CD47 expression, HSP90 expression and TUNEL in residual tumor tissue from each group at 30 d after surgery; scale bar, 200 µm. Blue: DAPI; red: PpIX; yellow: HSP90; green: TUNEL. H) Representative fluorescence images showing calcium influx and mitochondrial staining in live Cal33 cells from each group. Scale bar, 20 µm. Blue: DAPI; red: Rhod‐4 AM stain indicates calcium influx; green: MitoTracker Green FM stain indicates mitochondrial localization and membrane potential. Data are presented as the mean ± SD; n ≥ 3 independent experiments. p values were determined by two‐way ANOVA, Tukey's multiple‐comparison test (ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Recombinant human CD47 protein was obtained from MedChemExpress (MCE, USA).

Techniques: In Vivo, Irradiation, Staining, Immunohistochemical staining, Fluorescence, Expressing, TUNEL Assay, Membrane, Comparison

Journal: Nature Communications

Article Title: Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field

doi: 10.1038/s41467-023-37225-1

Figure Lengend Snippet: A Illustration of the assembly process and working principle of AMF-Bac. B The design of plasmids I and II. C The growth of Bac-HlpA/EGFP-BLPs and Bac-HlpA/EGFP after 42 °C treatment for the indicated times (0–120 min), as monitored by OD 600 . The data are shown as the mean ± SD (n = 3 independent experiments). D Bacteria from the different groups at 6 h in C were seeded on agar plates, and the number of colonies formed was counted. CFU, colony forming unit. The data are shown as the mean ± SD ( n = 3 independent experiments). E The lysis of Fe-Bac-HlpA/EGFP-BLPs after AMF treatment (310 kHz and 23.8 kA/m) for the indicated times (0–80 min), as observed by CLSM. The bacteria were tracked by their EGFP expression, and the Fe 3 O 4 NPs on the bacterial membrane were labeled with Cy5.5. Scale bar, 10 μm. F , G The lysis of engineered bacteria induced by AMF treatment in vivo. Mice bearing CT-26-luc xenografts were treated with engineered bacteria. The number of live bacteria in the tumor was measured by the spread plate method ( G ). The data ( G ) are shown as the mean ± SD ( n = 4 mice). H Optimization of the inducible expression conditions for overnight expression of CD47nb in the Fe-Bac-HlpA/CD47nb-BLPs. I The release of CD47nb (containing a Myc tag) from Fe-Bac-HlpA/CD47nb-BLPs upon 42 °C treatment for the indicated times (0–120 min) by western blot analysis using an anti-Myc tag antibody. J The release of CD47nb from Fe-Bac-HlpA/CD47nb-BLPs after AMF treatment (310 kHz and 23.8 kA/m) for the indicated times (0–160 min), as examined by dot blotting. K The affinity of the released CD47nb to the CD47 protein was verified by dot blotting. L Competition binding assay using the anti-CD47 antibody to examine the affinity of released CD47nb to CD47 in CT-26 cells. The data are shown as the mean ± SD ( n = 3 independent experiments). These experiments ( E , H – K ) were repeated three times independently with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant mouse CD47 protein (catalog No. 57231-MNAH) was purchased from Sino Biological Inc. (China).

Techniques: Lysis, Expressing, Labeling, In Vivo, Western Blot, Binding Assay

Journal: Nature Communications

Article Title: Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field

doi: 10.1038/s41467-023-37225-1

Figure Lengend Snippet: A Scheme and grouping of in vivo therapy ( n = 5). The colons of BALB/c mice were inoculated with CT-26-luc cells (1 × 10 5 cells/mouse) on day -7, and the mice were treated with the indicated engineered bacteria (1 × 10 8 CFU) by colon-specific administration on days 0 and 3, followed by AMF treatment (310 kHz and 23.8 kA/m) for 80 min at 24 h after colon administration. An anti-CD47 antibody (Anti-CD47ab; 20 mg/kg) and CD47nb (20 mg/kg) were i.p. injected on days 0 and 3 in G2 and G5, respectively. B Bioluminescence imaging to monitor tumor growth on days 0, 10, and 15. C Semi-quantitative results of the bioluminescence intensity of the tumor regions shown in panel B ( n = 5 mice). The bioluminescence intensities of each mouse at days 10 and 15 were normalized to day 0. D The number of metastatic tumors in the abdomen were counted on day 15 ( n = 5 mice). E , F The changes in red blood cell count (RBC; E ) and hemoglobin (HGB; F ) during therapy ( n = 5 mice). G Flow cytometry analysis of Anti-CD47ab (blue) or CD47nb (red) on RBCs in BALB/c mice bearing CT-26-luc colonic orthotopic xenografts after a single treatment with Anti-CD47ab or Fe-Bac-HlpA/CD47nb-BLPs + AMF. The Anti-CD47ab and CD47nb were detected using a fluorescein-labeled antibody against IgG and a nanobody, respectively. RBC without staining was used as the blank, and RBCs from healthy mice without treatments was used as control (Con). H Quantitative results of mean fluorescence intensity (MFI) in panel G ( n = 3 independent experiments). The data ( C – F , H ) are shown as the mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. * P < 0.05; ** P < 0.01 ; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Recombinant mouse CD47 protein (catalog No. 57231-MNAH) was purchased from Sino Biological Inc. (China).

Techniques: In Vivo, Injection, Imaging, Cell Counting, Flow Cytometry, Labeling, Staining, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: CD47 is a negative regulator of intestinal epithelial cell self-renewal following DSS-induced experimental colitis

doi: 10.1038/s41598-020-67152-w

Figure Lengend Snippet: CD47 upregulation in human intestinal epithelium from IBD patients. ( a ) CD47 immunostaining (red) and E-cadherin (green) doubling staining of colonic tissue section. Nuclei were counterstained with DAPI (blue) (n = 6, per). ( b ) Statistical analysis of CD47 expression in intestinal epithelium from non-IBD control (n = 6), patients with UC (n = 6) and patients with CD (n = 6). ***P < 0.001 versus non-IBD control.

Article Snippet: Colon tissues were frozen in OCT, and then sliced by Leica CM1950 at 6μm thickness, fixed in 4% paraformaldehyde, blocked with 10% FBS-PBS for 30 min, and incubated with anti-CD47 (R&D, BAF186) and anti-E-cadherin (Abcam, 76055) primary antibodies overnight at 4 °C.

Techniques: Immunostaining, Staining, Expressing

Journal: Scientific Reports

Article Title: CD47 is a negative regulator of intestinal epithelial cell self-renewal following DSS-induced experimental colitis

doi: 10.1038/s41598-020-67152-w

Figure Lengend Snippet: CD47 upregulation in mouse intestinal epithelium during DSS-induced experimental colitis. The mice (n = 20) were treated with 2%DSS for various time points. ( a ) CD47 immunostaining (red) and E-cadherin (green) doubling staining of colonic section in day 0, day 3, day 7, day 10 (n = 5). ( b,c ) Western blotting analysis of CD47 expression in dissected intestinal epithelium from DSS-treated mice on day 0, day 3, day 6, day 9 and day 12 post-treatment (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 versus day 0.

Article Snippet: Colon tissues were frozen in OCT, and then sliced by Leica CM1950 at 6μm thickness, fixed in 4% paraformaldehyde, blocked with 10% FBS-PBS for 30 min, and incubated with anti-CD47 (R&D, BAF186) and anti-E-cadherin (Abcam, 76055) primary antibodies overnight at 4 °C.

Techniques: Immunostaining, Staining, Western Blot, Expressing

Journal: Scientific Reports

Article Title: CD47 is a negative regulator of intestinal epithelial cell self-renewal following DSS-induced experimental colitis

doi: 10.1038/s41598-020-67152-w

Figure Lengend Snippet: CD47 upregulation in human IECs by inflammatory cytokine treatment. ( a,b ) Analysis of CD47 expression in HCoEpiC ( a ) and HT29 ( b ) cells with or without inflammatory cytokine treatment (100 ng/ml, 6 h). CD47 was labeled with anti-CD47 Abs followed by FITC. (c, d) Relative mRNA levels of CD47in HCoEpiC ( c ) and HT29 ( d ) cells with or without inflammatory cytokine treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control.

Article Snippet: Colon tissues were frozen in OCT, and then sliced by Leica CM1950 at 6μm thickness, fixed in 4% paraformaldehyde, blocked with 10% FBS-PBS for 30 min, and incubated with anti-CD47 (R&D, BAF186) and anti-E-cadherin (Abcam, 76055) primary antibodies overnight at 4 °C.

Techniques: Expressing, Labeling

Journal: Scientific Reports

Article Title: CD47 is a negative regulator of intestinal epithelial cell self-renewal following DSS-induced experimental colitis

doi: 10.1038/s41598-020-67152-w

Figure Lengend Snippet: CD47 deficiency protects the mice from DSS-induced experimental colitis. Mice (male, 20–22 g, n = 6) were given 2.5% DSS in drinking water for 7 day. ( a ) Bodyweight loss. ( b,c ) Colin length. ( d ) Disease activity index. ( e,f ) Histology of colon tissue (H&E staining) ( e ) and histological scoring ( f ). NS, no significance, ****P < 0.0001versus WT mice.

Article Snippet: Colon tissues were frozen in OCT, and then sliced by Leica CM1950 at 6μm thickness, fixed in 4% paraformaldehyde, blocked with 10% FBS-PBS for 30 min, and incubated with anti-CD47 (R&D, BAF186) and anti-E-cadherin (Abcam, 76055) primary antibodies overnight at 4 °C.

Techniques: Activity Assay, Staining

Journal: Scientific Reports

Article Title: CD47 is a negative regulator of intestinal epithelial cell self-renewal following DSS-induced experimental colitis

doi: 10.1038/s41598-020-67152-w

Figure Lengend Snippet: Negativecorrelation between the intestinal epithelial cell CD47 expression level and the cell proliferation capacity. ( a ) Organoids were stained with EDU (red). Nuclei were counterstained with DAPI (green). ( b ) EDU assay of proliferation of intestinal epithelial cells isolated from WT and CD47 −/− mice. Cells were treated with or without various inflammatory cytokines (n = 5). ***P < 0.001.

Article Snippet: Colon tissues were frozen in OCT, and then sliced by Leica CM1950 at 6μm thickness, fixed in 4% paraformaldehyde, blocked with 10% FBS-PBS for 30 min, and incubated with anti-CD47 (R&D, BAF186) and anti-E-cadherin (Abcam, 76055) primary antibodies overnight at 4 °C.

Techniques: Expressing, Staining, EdU Assay, Isolation

Journal: Scientific Reports

Article Title: CD47 is a negative regulator of intestinal epithelial cell self-renewal following DSS-induced experimental colitis

doi: 10.1038/s41598-020-67152-w

Figure Lengend Snippet: Intestinal epithelil cell CD47 inhibits cell wound healing and proliferation processes. ( a ) Overexpression or depletion of CT26 epithelial cells CD47 inhibits or promotes cell wound healing, respectively. ( b ) EDU assay of proliferation of CT26 cells. Cells were overexpressed or depleted CD47 via transfecting with CD47-expressing vector or CD47 siRNA. *P < 0.05, ***P < 0.001.

Article Snippet: Colon tissues were frozen in OCT, and then sliced by Leica CM1950 at 6μm thickness, fixed in 4% paraformaldehyde, blocked with 10% FBS-PBS for 30 min, and incubated with anti-CD47 (R&D, BAF186) and anti-E-cadherin (Abcam, 76055) primary antibodies overnight at 4 °C.

Techniques: Over Expression, EdU Assay, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: CD47 is a negative regulator of intestinal epithelial cell self-renewal following DSS-induced experimental colitis

doi: 10.1038/s41598-020-67152-w

Figure Lengend Snippet: CD47 deficiency upregulates the expression of OSKM in intestinal epithelial cells. ( a ) Western blot analysis of OSKM expression in intestinal epithelial cells from WT and CD47 −/− mice treated with 2.5% DSS. ( b ) Overexpression or depletion of CD47 in CT26 cells reduced or enhanced the expression of OSKM factors, respectively. ( c ) Depletion of CD47 in HT29 cells enhanced the expression of OSKM. ( d ) Exogenous TSP1 can downregulate expression of Sox2, Oct4 and cMyc in HT29 cell line, but not Klf4. NS, no significance, *P < 0.05, **P < 0.01, ****P < 0.0001 versus WT or control.

Article Snippet: Colon tissues were frozen in OCT, and then sliced by Leica CM1950 at 6μm thickness, fixed in 4% paraformaldehyde, blocked with 10% FBS-PBS for 30 min, and incubated with anti-CD47 (R&D, BAF186) and anti-E-cadherin (Abcam, 76055) primary antibodies overnight at 4 °C.

Techniques: Expressing, Western Blot, Over Expression

Journal: Scientific reports

Article Title: Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody.

doi: 10.1038/s41598-024-79865-3

Figure Lengend Snippet: Fig. 1. HX009 structure and in vitro characterization. (A) Molecular structure of HX009: 2 × 2 symmetric BsAb molecule; (B) HX009 binding to the recombinant CD47 proteins of different species including human, cynomolgus monkey (upper) and mouse (lower), binding EC50 (half maximal effective concentration) were determined per four-parameter equation fitting curves; (C) competitive binding to CD47 single-positive (upper panel) or CD47-PD1 double positive (lower panel) cells by HX009. Labeled B6H12 (anti-CD47) was competed out by SIRPα-Fc, but not by HX009 (weakened binding) in single-positive cell assay, while it was competed out in double-positive cell assays by both SIPRα-Fc and HX009. (D) T-cell activation luciferase report assays: upper panel: HX009 had enhanced T-cell activation (cis-binding) over HX008 (4X), whereas the enhancement is diminished by an anti-SIRPα neutralizing antibody but not by CD47-Fc (soluble CD47). Lower panel: there was no enhancement of T-cell activation by HX008 and SIRPα-Fc combo treatment due to trans-binding.

Article Snippet: By coupling the 7.5 μg/mL of HX009 antibody on the chip surface, the recombinant human (Atagenix), cynomolgus monkey (Acrobiosystems), mouse (Sinobiological) and rat (Sinobiological) CD47 proteins, at serial loading concentrations from 2 to 128 nM (two-fold dilution), or the recombinant human (Atagenix), cynomolgus monkey (Sinobiological), mouse (Sinobiological) and rat (Sinobiological) PD1 proteins at serial loading concentrations from 1 to 64 nM (two-fold dilution), flowed through the chip surface, a binding of 200-s to reach the target level above 770 RU, and a 20-min dissociation time with 10 mM of Glycine pH 1.7.

Techniques: In Vitro, Binding Assay, Recombinant, Concentration Assay, Labeling, Activation Assay, Luciferase

Journal: Scientific reports

Article Title: Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody.

doi: 10.1038/s41598-024-79865-3

Figure Lengend Snippet: Fig. 3. HX009 anti-tumor pharmacology studies in preclinical cancer models. (A) Pharmacology evaluation of HX009 in a humanized MC38-huCD47 mouse colon cancer model in huPD1-HuGEMM mice; (B) Pharmacology evaluation of HX009 in a humanized MC38-huCD47 mouse colon cancer model in huPD1 × huPD-L1 × huCD47 × huSIRPα-HuGEMM model; (C) Pharmacology evaluation of HX009 in three AML-PDX models as shown in the figures. Columns from Left to right: Leukemic burden was measured as percentage of human CD45+ cells in peripheral blood; survival was displayed as in Caplan-Meier plot; Leukemic loads in different organs at the termination, as % of human CD45+. SP spleen, BM bone marrow, PB peripheral blood. Bottom: The differential expressions of CD47 per IHC and RNAseq in three AML-PDX models are shown in the bottom table. Graphs in A-D showed mean tumor volume ± standard error of the mean (SEM). Significance was calculated using one-way ANOVA with post-hoc comparisons or Welch’s t- test between treatment groups and vehicle group. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Correlation of OX40 mRNA levels with HX009 anti-lymphoma activity represented by TGI (tumor growth inhibition) in DLBCL-PDX trial.

Article Snippet: By coupling the 7.5 μg/mL of HX009 antibody on the chip surface, the recombinant human (Atagenix), cynomolgus monkey (Acrobiosystems), mouse (Sinobiological) and rat (Sinobiological) CD47 proteins, at serial loading concentrations from 2 to 128 nM (two-fold dilution), or the recombinant human (Atagenix), cynomolgus monkey (Sinobiological), mouse (Sinobiological) and rat (Sinobiological) PD1 proteins at serial loading concentrations from 1 to 64 nM (two-fold dilution), flowed through the chip surface, a binding of 200-s to reach the target level above 770 RU, and a 20-min dissociation time with 10 mM of Glycine pH 1.7.

Techniques: Activity Assay, Inhibition