Journal: Molecular Oncology

Article Title: Improving the efficacy of osteosarcoma therapy: combining drugs that turn cancer cell ‘don't eat me’ signals off and ‘eat me’ signals on

doi: 10.1002/1878-0261.12556

Figure Lengend Snippet: Doxorubicin enhances the phagocytic efficacy of CD47 mAb in osteosarcomas. Schematic demonstration of macrophage‐mediated tumor phagocytosis: (A) Doxorubicin therapy: Doxorubicin induces calreticulin (CRT) on the surface of tumor cells, which ‘turns on’ an eat‐me signal and enables binding to an eat‐me receptor on macrophages. However, CD47 expression on tumor cells counteracts calreticulin‐mediated phagocytosis, (B) CD47 mAb therapy: CD47 mAb block the interaction of tumor CD47 with SIRPα and ‘turns off’ the don't eat‐me signal, (C) combined doxorubicin and CD47 mAb combination therapy synergized by turning the eat‐me signal ‘on’ and don't eat‐me signal ‘off’, and inducing macrophage‐mediated tumor cell phagocytosis. (D) Representative calreticulin staining of MNNG/HOS tumor cells treated with IgG, doxorubicin (0.5 μm), CD47 mAb (10 μg·mL −1 ), and combination therapy. (E) Corresponding quantitative area of calreticulin staining of control and treated tumor cells. (F) For phagocytosis assays, MNNG/HOS tumor cells were cocultured with murine bone marrow‐derived M1 macrophages for 6 h. Confocal images of CellBrite green‐labeled MNNG/HOS tumor cells and F4/80 + macrophages in the presence of different therapeutics. Cells exposed to combination therapy show an increased quantity of phagocytized tumor cells in macrophages (arrows; scale bar 10 μm) compared to monotherapy. (G) Corresponding relative phagocytosis, calculated as the number of macrophages with phagocytized cancer cell divided by total macrophages per five high‐power field × 100%. (H) Flow cytometry contour plots of M1 macrophages uptaking control IgG and doxorubicin plus CD47mAb‐treated MNNG/HOS tumor cells and (I) corresponding charts showing tumor cell phagocytosis in control and treated sets. Data are displayed as means ± SD of n = 5 experiments per group, P value as indicated, one‐way ANOVA.

Article Snippet: To evaluate macrophage‐mediated tumor phagocytosis (Mohanty et al ., ; Zhang et al ., ), in the presence of doxorubicin plus CD47 mAb combination therapy, MNNG/HOS osteosarcoma cells were labeled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine (CellBrite™ Green; Biotium, Fremont, CA, USA) according to the manufacturer's protocol and incubated at a 1 : 1 ratio with bone marrow‐derived M1 mouse macrophages in serum‐free IMDM, with 10 μg·mL −1 CD47 mAb, 500 nm doxorubicin, or both at 37 °C for 6 h. M1 polarization of macrophages was performed with previously established protocol (Mohanty et al ., ).

Techniques: Binding Assay, Expressing, Blocking Assay, Staining, Derivative Assay, Labeling, Flow Cytometry

Journal: Molecular Oncology

Article Title: Improving the efficacy of osteosarcoma therapy: combining drugs that turn cancer cell ‘don't eat me’ signals off and ‘eat me’ signals on

doi: 10.1002/1878-0261.12556

Figure Lengend Snippet: Ferumoxytol‐MRI shows increased T2 contrast in osteosarcomas after CD47 mAb combination therapy compared to monotherapy. (A) Schematic representation of experimental design: MNNG/HOS osteosarcoma cells were transfected with Tomato‐Td‐luciferase construct and implanted into the tibia of NSG mice ( n = 6/group). Tumor‐bearing mice were treated with CD47 mAb (10 mg·kg −1 , 3× per week) or doxorubicin (1 mg·kg −1 , 3× per week) or combination therapy. Five days after therapy, MRI was performed prior to and 24‐h post‐ferumoxytol administration (i.v.). (B) Representative T2‐weighted MR images of MNNG/HOS tumors before (upper row) and at 24 h after (lower row) intravenous injection of the macrophage marker ferumoxytol. Ferumoxytol enhancement is demonstrated by dark (negative) tumor enhancement on T2‐weighted MR images (red arrows). (C) T2 relaxation times of control and treated tumors. T2 relaxation times (quantitative measures of dark tumor ferumoxytol enhancement) were measured on T2 maps, which were generated based on multiecho T2 MSME sequences. All results are represented as mean ± SD from six tumors per experimental group, P ‐value as indicated, one‐way ANOVA.

Article Snippet: To evaluate macrophage‐mediated tumor phagocytosis (Mohanty et al ., ; Zhang et al ., ), in the presence of doxorubicin plus CD47 mAb combination therapy, MNNG/HOS osteosarcoma cells were labeled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine (CellBrite™ Green; Biotium, Fremont, CA, USA) according to the manufacturer's protocol and incubated at a 1 : 1 ratio with bone marrow‐derived M1 mouse macrophages in serum‐free IMDM, with 10 μg·mL −1 CD47 mAb, 500 nm doxorubicin, or both at 37 °C for 6 h. M1 polarization of macrophages was performed with previously established protocol (Mohanty et al ., ).

Techniques: Transfection, Luciferase, Construct, Injection, Marker, Generated

Journal: Molecular Oncology

Article Title: Improving the efficacy of osteosarcoma therapy: combining drugs that turn cancer cell ‘don't eat me’ signals off and ‘eat me’ signals on

doi: 10.1002/1878-0261.12556

Figure Lengend Snippet: Histopathology shows M1 macrophage activation after doxorubicin and CD47 mAb therapy. (A) Representative Prussian blue‐DAB (scale bar 50 μm) iron stains and immunofluorescent F4/80 confocal images (scale bar 50 μm) of MNNG/HOS tumors show increasing quantities of iron oxide nanoparticles and macrophages in tumors treated with control Ab, CD47 mAb, doxorubicin, and combination therapies. (B) Corresponding quantitative area of Prussian blue‐DAB and F4/80‐positive macrophages in control and treated tumors. (C) Confocal immunofluorescent images and (D) Corresponding quantitative area of CD80, iNOS, and calreticulin staining in control and treated tumors (scale bar 50 μm). All results are represented as mean ± SD from six tumors per experimental group, P ‐value as indicated, one‐way ANOVA.

Article Snippet: To evaluate macrophage‐mediated tumor phagocytosis (Mohanty et al ., ; Zhang et al ., ), in the presence of doxorubicin plus CD47 mAb combination therapy, MNNG/HOS osteosarcoma cells were labeled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine (CellBrite™ Green; Biotium, Fremont, CA, USA) according to the manufacturer's protocol and incubated at a 1 : 1 ratio with bone marrow‐derived M1 mouse macrophages in serum‐free IMDM, with 10 μg·mL −1 CD47 mAb, 500 nm doxorubicin, or both at 37 °C for 6 h. M1 polarization of macrophages was performed with previously established protocol (Mohanty et al ., ).

Techniques: Histopathology, Activation Assay, Staining

Journal: Molecular Oncology

Article Title: Improving the efficacy of osteosarcoma therapy: combining drugs that turn cancer cell ‘don't eat me’ signals off and ‘eat me’ signals on

doi: 10.1002/1878-0261.12556

Figure Lengend Snippet: Bioluminescence imaging shows decreased tumor growth of osteosarcomas after doxorubicin and CD47 mAb combination therapy. (A) Bioluminescent in vivo images of mice with intratibial MNNG/HOS osteosarcomas before and after therapy with IgG, CD47 mAb, doxorubicin, and combination therapy. (B) Total quantified flux of MNNG/HOS osteosarcomas at different time points after intravenous treatment with IgG, doxorubicin, CD47 mAb, or combination therapy. Results are represented as mean ± SD from six tumors per experimental group, P ‐value as indicated, one‐way ANOVA.

Article Snippet: To evaluate macrophage‐mediated tumor phagocytosis (Mohanty et al ., ; Zhang et al ., ), in the presence of doxorubicin plus CD47 mAb combination therapy, MNNG/HOS osteosarcoma cells were labeled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine (CellBrite™ Green; Biotium, Fremont, CA, USA) according to the manufacturer's protocol and incubated at a 1 : 1 ratio with bone marrow‐derived M1 mouse macrophages in serum‐free IMDM, with 10 μg·mL −1 CD47 mAb, 500 nm doxorubicin, or both at 37 °C for 6 h. M1 polarization of macrophages was performed with previously established protocol (Mohanty et al ., ).

Techniques: Imaging, In Vivo

Journal: Molecular Oncology

Article Title: Improving the efficacy of osteosarcoma therapy: combining drugs that turn cancer cell ‘don't eat me’ signals off and ‘eat me’ signals on

doi: 10.1002/1878-0261.12556

Figure Lengend Snippet: Doxorubicin plus CD47 mAb combination therapy prevents pulmonary metastasis in osteosarcoma‐bearing mice. (A) Low‐power (10×, H&E stain) view of the lungs showed metastasis from the primary tumor. (B) Corresponding summed tumor area of pulmonary metastases in mice treated with control IgG, doxorubicin, CD47 mAb, and combination therapy. Results are represented as mean ± SD from six animals per experimental group, P ‐value as indicated, one‐way ANOVA.

Article Snippet: To evaluate macrophage‐mediated tumor phagocytosis (Mohanty et al ., ; Zhang et al ., ), in the presence of doxorubicin plus CD47 mAb combination therapy, MNNG/HOS osteosarcoma cells were labeled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine (CellBrite™ Green; Biotium, Fremont, CA, USA) according to the manufacturer's protocol and incubated at a 1 : 1 ratio with bone marrow‐derived M1 mouse macrophages in serum‐free IMDM, with 10 μg·mL −1 CD47 mAb, 500 nm doxorubicin, or both at 37 °C for 6 h. M1 polarization of macrophages was performed with previously established protocol (Mohanty et al ., ).

Techniques: Staining

Journal: Molecular Oncology

Article Title: Improving the efficacy of osteosarcoma therapy: combining drugs that turn cancer cell ‘don't eat me’ signals off and ‘eat me’ signals on

doi: 10.1002/1878-0261.12556

Figure Lengend Snippet: Doxorubicin plus CD47 mAb combination therapy improves survival in osteosarcoma‐bearing mice. (A) Kaplan–Meier survival curves demonstrate a significant survival benefit of combination therapy as compared to control, doxorubicin, and anti‐CD47 alone, log‐rank Mantel–Cox test (log‐rank Mantel–Cox test). (B) Tumor T2 relaxation times, obtained from ferumoxytol‐MRI, correlate with survival outcomes of mice receiving combination therapy ( r = −0.9, P = 0.01, Spearman rank correlation, n = 6).

Article Snippet: To evaluate macrophage‐mediated tumor phagocytosis (Mohanty et al ., ; Zhang et al ., ), in the presence of doxorubicin plus CD47 mAb combination therapy, MNNG/HOS osteosarcoma cells were labeled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine (CellBrite™ Green; Biotium, Fremont, CA, USA) according to the manufacturer's protocol and incubated at a 1 : 1 ratio with bone marrow‐derived M1 mouse macrophages in serum‐free IMDM, with 10 μg·mL −1 CD47 mAb, 500 nm doxorubicin, or both at 37 °C for 6 h. M1 polarization of macrophages was performed with previously established protocol (Mohanty et al ., ).

Techniques:

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: ( a ). Cytoscape network visualization of the genes which are significantly correlated with CD47 expression in both human and murine atherosclerotic plaque reveals a high number of TNF-α-related factors (indicated in blue), including ligands, receptors, and downstream signaling factors. ( b ). PANTHER pathway analysis of those genes which were (a) significantly associated with CD47 expression in mouse and human vascular tissue and (b) have been previously associated with atherosclerosis through the STAGE study , identifies “ inflammation mediated by chemokine and cytokine signaling pathway ” as the most over-abundant pathway associated with CD47 expression in vascular tissue. ( c ). Using the Hybrid Mouse Diversity Panel (HMDP), which correlates aortic gene expression with Luminex cytokine array data of plasma samples from over 100 inbred strains of mice, we found that vascular CD47 expression is positively correlated with three inflammatory cytokines in vivo, including TNF-α, IL-2 and CXCL1. Correlation data shown for CD47 and TNF-α. ( d ). Co-expression studies confirm that TNF-α and CD47 expression are positively correlated in human carotid endarterectomy samples from the BiKE validation study. The Pearson correlation coefficient was determined assuming a Gaussian distribution and P values were determined using a two-tailed test. ( e ). Experiments with primarily cultured mouse aortic SMCs indicate that TNF-α reproducibly induces CD47 mRNA upregulation, while a number of other classical pro-atherosclerotic stimuli have no significant effect. Notably, CXCL1, IL4, TGFβ and IL-2 fail to induce CD47 expression in vitro, as assessed by ANOVA. ( f ). Additional studies suggest that the effect of TNF-α on CD47 expression persists in the presence of oxidized LDL, as occurs in the atherosclerotic plaque. ( g ). Western blotting confirms that TNF-α induces CD47 expression in vascular cells at the protein level. For gel source data, see . ( h ). Immunocytochemistry studies of HCASMCs confirm that CD47 expression is induced on the cell surface of TNF-α treated cells. TNF-α effect is assessed by co-staining for HMGB1, and antibody specificity is confirmed with isotype control and recombinant CD47 peptide quenching assays. ( i ). Multiple assays (including FACS, Taqman and immunocytochemistry studies) reveal that CD47 expression is downregulated on vascular SMCs during programmed cell death, as has previously been observed with inflammatory cells. ( j ). Confirmatory assays in cultured human coronary artery SMC reveal that TNF-α induces changes similar to those observed in murine cells , including an induction of CD47 under physiological conditions and a blunting of its expected downregulation during apoptosis. ( k ). TNF-α’s capacity to impair CD47 downregulation during programmed cell death is also observed in mouse SMCs simultaneously exposed to pro-apoptotic stimuli and oxidized LDL. ( l ). No correlation between CD47 and other candidate cytokines was observed in the BiKE biobank, further supporting a specific relationship between CD47 and TNF-α. ( m ). Representative FACS-based apoptosis panels from cells exposed to the conditions used in confirm that TNF-α suppresses efferocytosis despite increasing programmed cell death. Comparisons made by two-tailed t tests, unless otherwise specified. *** = P < 0.001, * = P < 0.05. Error bars represent the SEM.

Article Snippet: In some experiments, membranes loaded with protein were incubated with anti-CD47 Ab that had been preabsorbed with CD47 peptide (R&D systems, 1866-CD, 1:5 dilution) for 16 hours, to determine the specificity of the primary Ab.

Techniques: Expressing, Gene Expression, Luminex, Clinical Proteomics, In Vivo, Biomarker Discovery, Two Tailed Test, Cell Culture, In Vitro, Western Blot, Immunocytochemistry, Staining, Control, Recombinant

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: In vivo serological data and additional in silico and bioinformatic data ( a ). Complete serological studies (including blood count, liver function studies, basic metabolic panel, and fasting glucose) from the 4 week apoE −/− -AngII atherosclerosis model indicate that anti-CD47 Ab induces a significant reduction in hemoglobin and compensatory reticulocytosis, consistent with prior reports 4 , 7 . The erythrophagocytosis of senescent RBCs appears to be self-limited, and no anemia was observed in the chronic atherosclerosis model or the reduced dose model (P = 0.54 and 0.57, respectively). No significant difference in any other serum marker is observed except for an increase in serum creatinine, which does not deviate outside of the reference range. Metabolic parameters and leukocyte differential data from the 12 week chronic atherosclerosis model are displayed at the bottom of the table. ( b ). Additional Upstream Regulator Analysis (URA) bioinformatic analyses of the Cytoscape data displayed in Extended Data Figure 7a performed within the Ingenuity Pathway Analysis (IPA) software identifies a number of TNF-α related factors (indicated in red) which are predicted to mediate transcriptional regulatory roles in the gene network shown in that panel. P-values were determined from Fisher’s Exact Test by comparing overlap of co-expressed genes with known upstream regulators from the Ingenuity Knowledge Base. ( c ). Several additional DAVID -based bioinformatics analyses including ( KEGG , SMART , PANTHER and GO analyses) confirm the association between CD47 and inflammatory signaling related to the TNF-α pathway (indicated in red). Blue panels indicate the –logp10 value for each identified factor. ( d ). Transcription factor binding site prediction algorithms identify several putative NFKB family binding sites within the CD47 promoter, as displayed in Extended Data Figure 8a . ( d ). List of primers used in this study.

Article Snippet: In some experiments, membranes loaded with protein were incubated with anti-CD47 Ab that had been preabsorbed with CD47 peptide (R&D systems, 1866-CD, 1:5 dilution) for 16 hours, to determine the specificity of the primary Ab.

Techniques: In Vivo, In Silico, Marker, Software, Binding Assay

Journal: Journal of Nanobiotechnology

Article Title: Multifunctional exosome-mimetics for targeted anti-glioblastoma therapy by manipulating protein corona

doi: 10.1186/s12951-021-01153-3

Figure Lengend Snippet: Study design and characterization of Ang-EM. A Illustration of DTX@Ang-EM preparation, avoiding protein corona formation, escaping phagocytosis, BBB penetration and GBM targeting. B Size distribution and zeta potential of Lipo, Ang-Lipo and Ang-EM. C TEM images of Lipo, Ang-Lipo and Ang-EM. Scale bar = 100 nm. D Summary and comparison of mean size, polymey distribution index and zeta potential of Lipo, Ang-Lipo and Ang-EM. E SDS-page analysis of protein profiles of Ang-EM. MP, membrane proteins; Exo, Exosomes; cyto, cytosolic proteins. F Western blot of protein markers of CD63, CD9 and CD47 on EM and Exo

Article Snippet: The presence of protein markers CD47 (ab175388, Abcam, UK), CD9 (ab92726, Abcam, UK) and CD63 (ab216130, Abcam, UK) were detected via western blotting and analyzed using a gel imaging system (ChemiDocTM Touch, Bio-Rad, USA).

Techniques: Zeta Potential Analyzer, Comparison, SDS Page, Membrane, Western Blot

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: ( a ). Cytoscape network visualization of the genes which are significantly correlated with CD47 expression in both human and murine atherosclerotic plaque reveals a high number of TNF-α-related factors (indicated in blue), including ligands, receptors, and downstream signaling factors. ( b ). PANTHER pathway analysis of those genes which were (a) significantly associated with CD47 expression in mouse and human vascular tissue and (b) have been previously associated with atherosclerosis through the STAGE study , identifies “ inflammation mediated by chemokine and cytokine signaling pathway ” as the most over-abundant pathway associated with CD47 expression in vascular tissue. ( c ). Using the Hybrid Mouse Diversity Panel (HMDP), which correlates aortic gene expression with Luminex cytokine array data of plasma samples from over 100 inbred strains of mice, we found that vascular CD47 expression is positively correlated with three inflammatory cytokines in vivo, including TNF-α, IL-2 and CXCL1. Correlation data shown for CD47 and TNF-α. ( d ). Co-expression studies confirm that TNF-α and CD47 expression are positively correlated in human carotid endarterectomy samples from the BiKE validation study. The Pearson correlation coefficient was determined assuming a Gaussian distribution and P values were determined using a two-tailed test. ( e ). Experiments with primarily cultured mouse aortic SMCs indicate that TNF-α reproducibly induces CD47 mRNA upregulation, while a number of other classical pro-atherosclerotic stimuli have no significant effect. Notably, CXCL1, IL4, TGFβ and IL-2 fail to induce CD47 expression in vitro, as assessed by ANOVA. ( f ). Additional studies suggest that the effect of TNF-α on CD47 expression persists in the presence of oxidized LDL, as occurs in the atherosclerotic plaque. ( g ). Western blotting confirms that TNF-α induces CD47 expression in vascular cells at the protein level. For gel source data, see . ( h ). Immunocytochemistry studies of HCASMCs confirm that CD47 expression is induced on the cell surface of TNF-α treated cells. TNF-α effect is assessed by co-staining for HMGB1, and antibody specificity is confirmed with isotype control and recombinant CD47 peptide quenching assays. ( i ). Multiple assays (including FACS, Taqman and immunocytochemistry studies) reveal that CD47 expression is downregulated on vascular SMCs during programmed cell death, as has previously been observed with inflammatory cells. ( j ). Confirmatory assays in cultured human coronary artery SMC reveal that TNF-α induces changes similar to those observed in murine cells , including an induction of CD47 under physiological conditions and a blunting of its expected downregulation during apoptosis. ( k ). TNF-α’s capacity to impair CD47 downregulation during programmed cell death is also observed in mouse SMCs simultaneously exposed to pro-apoptotic stimuli and oxidized LDL. ( l ). No correlation between CD47 and other candidate cytokines was observed in the BiKE biobank, further supporting a specific relationship between CD47 and TNF-α. ( m ). Representative FACS-based apoptosis panels from cells exposed to the conditions used in confirm that TNF-α suppresses efferocytosis despite increasing programmed cell death. Comparisons made by two-tailed t tests, unless otherwise specified. *** = P < 0.001, * = P < 0.05. Error bars represent the SEM.

Article Snippet: Antibody specificity was confirmed using isotype control and by preincubating the anti-CD47 Ab with recombinant CD47 antigen (R&D Systems) in a 1:5 ratio for 16h at 4°C before application.

Techniques: Expressing, Gene Expression, Luminex, Clinical Proteomics, In Vivo, Biomarker Discovery, Two Tailed Test, Cell Culture, In Vitro, Western Blot, Immunocytochemistry, Staining, Control, Recombinant

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: In vivo serological data and additional in silico and bioinformatic data ( a ). Complete serological studies (including blood count, liver function studies, basic metabolic panel, and fasting glucose) from the 4 week apoE −/− -AngII atherosclerosis model indicate that anti-CD47 Ab induces a significant reduction in hemoglobin and compensatory reticulocytosis, consistent with prior reports 4 , 7 . The erythrophagocytosis of senescent RBCs appears to be self-limited, and no anemia was observed in the chronic atherosclerosis model or the reduced dose model (P = 0.54 and 0.57, respectively). No significant difference in any other serum marker is observed except for an increase in serum creatinine, which does not deviate outside of the reference range. Metabolic parameters and leukocyte differential data from the 12 week chronic atherosclerosis model are displayed at the bottom of the table. ( b ). Additional Upstream Regulator Analysis (URA) bioinformatic analyses of the Cytoscape data displayed in Extended Data Figure 7a performed within the Ingenuity Pathway Analysis (IPA) software identifies a number of TNF-α related factors (indicated in red) which are predicted to mediate transcriptional regulatory roles in the gene network shown in that panel. P-values were determined from Fisher’s Exact Test by comparing overlap of co-expressed genes with known upstream regulators from the Ingenuity Knowledge Base. ( c ). Several additional DAVID -based bioinformatics analyses including ( KEGG , SMART , PANTHER and GO analyses) confirm the association between CD47 and inflammatory signaling related to the TNF-α pathway (indicated in red). Blue panels indicate the –logp10 value for each identified factor. ( d ). Transcription factor binding site prediction algorithms identify several putative NFKB family binding sites within the CD47 promoter, as displayed in Extended Data Figure 8a . ( d ). List of primers used in this study.

Article Snippet: Antibody specificity was confirmed using isotype control and by preincubating the anti-CD47 Ab with recombinant CD47 antigen (R&D Systems) in a 1:5 ratio for 16h at 4°C before application.

Techniques: In Vivo, In Silico, Marker, Software, Binding Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) Existing model by which SIRPα suppresses phagocytosis by interacting in trans with CD47 on target cells. See text for details. The 3 Ig-like domains of SIRPα (1 IgV and 2 IgCs) and the single Ig-V domain of CD47 are shown as ellipses. Mβs, macrophages. ( B ) Depiction of SIRPα variants and their functional characteristics. SIRPα FFFF contained substitution of tyrosine (Y)-to-phenylalanine (F) substitution at Y436, 460, 477, and 501; SIRPα ΔIC lacked most of the cytoplasmic domain of SIRPα, ending with arginine 401; SIRPα T96V carried a threonine (T)-to-valine (V) mutation at position 96 (shown by lavender star), which abolishes CD47-binding; SIRPα T96V,FFFF had the T96V and FFFF mutations; SIRPα T96V,ΔIC had the T96V and the ΔIC mutations. KO, knock-out. ITIM, immunoreceptor tyrosine-based inhibitory motif. ( C to G ) SIRPα variants or empty vector were expressed in SIRPα KO BMDMs and tested. Wild-type (WT) BMDMs were used as control. ( C ) Schematic representation of assays performed. Fc, fragment crystallizable. ( D ) Flow cytometry analyses of SIRPα expression and CD47-binding. APC, allophycocyanin. AF647, Alexa fluor 647. ( E and F ) Representative ( E ) and compiled data ( F ) of pHrodo-based phagocytosis assays using L1210 derivatives expressing Tac and opsonized with Tac monoclonal antibody (mAb) 7G7, as targets. Positive cells with percentages are boxed. G , Efficiency of phagocytosis inhibition in SIRPα KO BMDMs expressing or not the indicated SIRPα variants was calculated using the values in ( F ). SIRPα KO expressing WT SIRPα or empty vector displayed 100% and 0% inhibition efficiency, respectively. All data are means ± s.e.m., **** p < 0.0001. Results in ( D and E ) are representative of 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that are representative of 3 experiments. Results in ( F and G ) are pooled from a total of 6 mice studied in 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that involved 3 mice in 3 experiments. Each symbol in ( F ) represents one mouse.

Article Snippet: A histidine (His)-tagged version of mouse CD47 (CD47-His; Cat# 57231-M08H) was from Sino Biological (Beijing, China).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: A histidine (His)-tagged version of mouse CD47 (CD47-His; Cat# 57231-M08H) was from Sino Biological (Beijing, China).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: A histidine (His)-tagged version of mouse CD47 (CD47-His; Cat# 57231-M08H) was from Sino Biological (Beijing, China).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis

Journal: Journal of Pharmaceutical Analysis

Article Title: Dual-targeted halofuginone hydrobromide nanocomplexes for promotion of macrophage repolarization and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes in adjuvant-induced arthritis in rats

doi: 10.1016/j.jpha.2024.100981

Figure Lengend Snippet: Function and cellular uptake of hybrid membrane (M). (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the membrane proteins of marker, red blood cell membrane (RBCm), macrophage membrane (Møm), M, and hyaluronic acid (HA)-modified hybrid membrane (M)-camouflaged poly lactic- co -glycolic acid (PLGA) loaded halofuginone hydrobromide (HF) nanoparticles (NPs) (HA-M@P@HF NPs). (B) Detection of CD11b, CD47, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in HA-M@P@HF NPs. (C) Fluorescent images of P@chlorin e6 (Ce6) and HA-M@P@Ce6 uptake in RAW264.7 cells, activated macrophages, human fibroblast-like synoviocytes (HFLS), HFLS-rheumatoid arthritis (RA), and human umbilical vein endothelial cell (HUVEC) for 4 h. (D) Cumulative release of HF from HA-M@P@HF NPs at pH 5.4 and 7.4. (E, F) Release kinetics of HA-M@P@HF NPs and various mathematical models of release mechanisms (i.e., zero-order model, first-order model, Higuchi model, Peppas model, and Weibull model) at pH 5.4 (E) and 7.4 (F). Data are presented as mean ± standard deviation ( n = 3). ∗∗∗ P < 0.001. MFI: mean fluorescence intensity.

Article Snippet: We also utilized antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), P53, P21, Bcl-2-associated X protein (BAX), Bcl-2, CD80, CD206, Ki67, CD44, CD11b, and CD47, obtained from Proteintech Group Inc. (Wuhan, China).

Techniques: Membrane, Polyacrylamide Gel Electrophoresis, SDS Page, Marker, Modification, Standard Deviation, Fluorescence

Journal: PLoS Biology

Article Title: Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation

doi: 10.1371/journal.pbio.2006859

Figure Lengend Snippet: ( a ) CX3CR1 GFP/+ mice were injected with CpG-C or vehicle, and 24 hours later mRNA expression levels in sorted microglia cells were quantified using RT-qPCR. In one experiment, six animals of each group were pooled into a single sample, and in the second experiment, two CpG-C–treated animals and three controls were analyzed separately ( n = 3–4 from 8–9 animals). As expected, Tmem119 , a general microglia marker, was unaffected by the treatment ( t (5) = 0.371, p = 0.7258). ( b ) The death ligands, Tnfsf10 and Fasl , were elevated by 3–4-fold by a single CpG-C injection ( t (5) = 2.564, p = 0.0437; and t (5) = 2.36, p = 0.0324, respectively). ( c ) Expression levels of receptors related to phagocytosis were significantly higher in microglia of CpG-C–treated animals. While no change was apparent in Cd36 ( t (5) = 0.3966, p = 0.7080) and Cd68 ( t (5) = 0.01655, p = 0.9874), a significant increase was evident in Cd47 ( t (5) = 2.819, p = 0.0186), Trem2 ( t (5) = 2.762, p = 0.0199), and Marco (which was not detected in control animals) ( t (4) = 4.499, p = 0.0108). ( d ) While RNA of the inflammatory cytokines Il-6 and Il1-β was not affected by CpG-C treatment ( t (5) = 0.04089, p = 0.9690; t (5) = 0.4417, p = 0.6772, respectively), Tnf ( t (4) = 3.207, p = 0.0163) and Inf-γ ( t (4) = 2.394, p = 0.0374), which synergistically induce apoptosis in tumor cells , and Nos2 ( t (5) = 2.744, p = 0.0203), which is tumoricidal at high concentrations , were increased following CpG-C injection. Data are presented as mean (±SEM). The underlying data for this figure can be found in , and our gating strategies are provided in . Cd , cluster of differentiation; Fasl , Fas ligand; GFP, green fluorescent protein; Il , interleukin; Inf-γ , interferon gamma; Marco , macrophage receptor with collagenous structure; Nos2 , nitric oxide synthase 2; RT-qPCR, real-time quantitative polymerase chain reaction; Tmem119 , transmembrane protein 119; Tnf , tumor necrosis factor; Tnfsf10 , tumor necrosis factor superfamily member 10; Trem2 , triggering receptor expressed on myeloid cells 2.

Article Snippet: All primers and probes were purchased from Applied Biosystems, Cd36 (Mm00432403_m1), Cd47 (Mm00495006_m1), Cd68 (Mm03047343_m1), Fas ligand ( Fasl ) (Mm00438864_m1), Gapdh (Mm99999915_g1), interferon gamma ( Inf-γ ) (Mm01168134_m1), interleukin ( Il ) 1-β (Mm00434228_m1), Il-6 ( Mm00446190_m1) , Macrophage receptor with collagenous structure ( Marco ) (Mm00440250_m1), nitric oxide synthase 2 ( Nos2 ) (Mm00440502_m1) , transmembrane protein 119 ( Tmem119 ) (Mm00525305_m1), tumor necrosis factor ( Tnf ) (Mm00443258_m1), tumor necrosis factor superfamily member 10 ( Tnfsf10 ) (Mm01283606_m1), and triggering receptor expressed on myeloid cells 2 ( Trem2 ) (Mm04209424_g1).

Techniques: Injection, Expressing, Quantitative RT-PCR, Marker, Control, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation

doi: 10.1371/journal.pbio.2006859

Figure Lengend Snippet: Systemic prophylactic treatment with CpG-C during the perioperative period activates microglia to induce apoptosis in tumor cells and phagocytize them, resulting in reduced brain metastases colonization. A few weeks to months may pass from the time of cancer diagnosis to the time of primary tumor excision . During this period, and a few weeks after surgical excision (known as the perioperative period), there is a high risk for developing brain metastasis with terminal consequences. CpG-C, a TLR9 agonist, given as a systemic prophylactic treatment during this crucial period, infiltrates the brain and activates microglia (1), increasing their expression of Tnfsf10 and Fasl , resulting in contact-dependent induced apoptosis of tumor cells (2). Furthermore, Cd47 , Trem2 , and Marco expression is increased, triggering enhanced microglial phagocytosis and dismantling of tumor cells (3), thereby reducing brain metastasis colonization. CD, cluster of differentiation; FasL, Fas ligand; Marco, macrophage receptor with collagenous structure; TLR9, toll-like receptor 9; Tnfsf10, tumor necrosis factor superfamily member 10; TREM2, triggering receptor expressed on myeloid cells 2.

Article Snippet: All primers and probes were purchased from Applied Biosystems, Cd36 (Mm00432403_m1), Cd47 (Mm00495006_m1), Cd68 (Mm03047343_m1), Fas ligand ( Fasl ) (Mm00438864_m1), Gapdh (Mm99999915_g1), interferon gamma ( Inf-γ ) (Mm01168134_m1), interleukin ( Il ) 1-β (Mm00434228_m1), Il-6 ( Mm00446190_m1) , Macrophage receptor with collagenous structure ( Marco ) (Mm00440250_m1), nitric oxide synthase 2 ( Nos2 ) (Mm00440502_m1) , transmembrane protein 119 ( Tmem119 ) (Mm00525305_m1), tumor necrosis factor ( Tnf ) (Mm00443258_m1), tumor necrosis factor superfamily member 10 ( Tnfsf10 ) (Mm01283606_m1), and triggering receptor expressed on myeloid cells 2 ( Trem2 ) (Mm04209424_g1).

Techniques: Biomarker Discovery, Expressing

Journal: bioRxiv

Article Title: Isoform switch of CD47 provokes macrophage-mediated pyroptosis in ovarian cancer

doi: 10.1101/2025.04.17.649282

Figure Lengend Snippet: (A) Diagram depicting the workflow for identifying immune checkpoints undergoing isoform switching in ovarian cancer. Long reads and short reads transcriptome data were used to analyze isoform switches. (B) Venn diagram indicates 7 dysregulated immune checkpoint ligands were detected isoform switch comparing SOCs with FTs. (C) Volcano plot of differential expression of immune checkpoints in TCGA-OV and GTEx-ovary. (D) Representative genomic visualization and schematic diagram of gene structure of CD47 based on long reads sequencing of the FT and SOC sample. (E) Representative genomic visualization of CD47 in FT, ovary, peritoneum, and SOC tissues. Junction between exons were shown as arc. (F) PSI index of CD47 exon 9 and 10 skipping in TCGA-OV and GTEx-ovary. (G) SpliZ value of epithelial cells in scRNA-seq data from FT and SOCs. (H) Isoform percentage of CD47-L and CD47-S in TCGA-OV and GTEx ovary database. (I and J) Genomic visualization (I) and quantification (J) of MicroAS-seq targeting CD47 micro-exon 9 and 10 skipping regions of FT and SOC samples. (K) Representative images of RNA FISH of CD47 isoforms detected by immunohistochemistry in TMA containing 144 FT and 394 SOC spots. (L) Normalized relative fluorescence intensity of CD47-L and CD47-S of samples in TMA. (M) Representative images of immunohistochemistry on the same microarray using anti-CD47 antibody. (N) Proportion of the CD47-L isoform in FTs (n=10) and SOCs (n=28) calculated based on peptide abundance from quantitative proteomics. (O) The ascites volume of patients with ovarian cancer in CD47-L and CD47-S dominant groups. (P) Prognostic analysis of the correlation between CD47 isoforms and overall survival of Qilu cohort. 198 patients were divided into PSI low (n=36) and high (n=162) group. (Q) Kaplan-Meier survival curves showing overall survival probability stratified by CD47 PSI expression levels (high or low) and BRCA mutation status (wild-type or mutant). The p value was obtained by log-rank test.

Article Snippet: GFP-CD47-L, a plasmid coding GFP tagged CD47 with its own short 3’UTR, were purchased from Addgene (GFP_CD47_SU, #65473).

Techniques: Quantitative Proteomics, Sequencing, Immunohistochemistry, Fluorescence, Microarray, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Isoform switch of CD47 provokes macrophage-mediated pyroptosis in ovarian cancer

doi: 10.1101/2025.04.17.649282

Figure Lengend Snippet: (A) Flowchart illustrating the CD47 isoform characteristics at the single-cell and spatial levels in relation to cell-cell communication. Spatial isoform characteristics were validated using multicolor fluorescence staining and RNA FISH. (B) Uniform manifold approximation and projection (UMAP) embedding of cells from 20 ovarian cancer samples, colored by major cell types. Epithelial cell group was subclustered into CD47-L and CD47-S cells based on the SpliZ value. (C) UMAP projection of CD47 expression level. Dashed circles indicate epithelial cell subgroup. (D) Heatmap of interaction strength between CD47-L/S epithelial subgroups and other cells via CD86, ICAM, ITGB2, and CD40 signaling. (E) Stacked bar plot indicated the global interaction strength of CD47-L and CD47-S subgroups with other cell types. (F) Bubble diagram indicating the enriched pathway of CD47-L/S epithelial cell subgroups and other cell types. (G) Cell-cell communication network illustrating interactions between myeloid cells, fibroblasts, endothelial cells, NKT cells, B cells, pre-B cells, and epithelial cells in CD47-L/S dominant samples. The thickness of the connections represents the strength of the interactions. (H) Dot plot showing the communication probability (Commun. Prob.) of ligand-receptor interactions between M-Epi and NKT-Epi in CD47-S/L dominant samples. The color gradient represents communication strength, ranging from low (blue) to high (red), and dot size indicates interaction significance. (I, J) Normalized RNA expression of T cell activation markers (I) and pro-inflammatory markers of macrophage (J) . Blue, CD47-L epithelial cell subgroup; red, CD47-S epithelial cell subgroup. (K) Myeloid subgroups proportions in CD47-L/S dominant samples. Pro-inflammatory M1 score and immunosuppressive M2 score of each myeloid subgroups were annotated on the right. (L) Cell communication network of epithelial cells and macrophages cross the SP8 tissue section. Lines mark the nearest tumor cells adjacent to each macrophage. Colors represent SpliZ values in epithelial cells. Dashed lines outline L-dominant epithelial cell region. (M) Distance between macrophage and nearest epithelial CD47-L/S spots (up). Distance of the epithelial spot from its nearest neighbor in the CD47-L/S group (down). (N) Spatial distribution map of CD47 SpliZ value calculated based on 3’ sequencing, long reads sequencing, and scMicroAS-seq data from same spatial transcriptome cDNA library of OVCST tissue. Each hexagon represents a spatial spot, with color intensity reflecting markers levels. Dashed lines outline epithelial cell region. (O) Macrophage infiltration score (Up) and CD47 SpliZ value (Down) in Epithelial cells A, B, C, D subgroups. (P) Multiple IHC and RNA FISH image of T1 tissue. Epithelial marker (PANCK, magenta), macrophage markers (CD68, cyan; HLA-DR, yellow; CD163, green), smooth muscle marker (SMA, red), and nuclear stain (DAPI, blue). Heatmap of normalized S-L value determined by florescence intensity of RNA FISH in T1 tissue (24×24 bins). Red square, CD47-S ROI; blue, square, CD47-L ROI (right). Dashed lines outline epithelial cell region. (Q) RNA FISH (first column) and immunofluorescence (second and third column) images of corresponding CD47-L ROI-1 and CD47-S ROI-1. (R) Normalized intensity density of DAPI, PANCK, SMA, CD68, HLA, and CD163 in CD47-L (blue, n=8) and CD47-S (red, n=13) dominant regions. Intensity density was normalized to DAPI density.

Article Snippet: GFP-CD47-L, a plasmid coding GFP tagged CD47 with its own short 3’UTR, were purchased from Addgene (GFP_CD47_SU, #65473).

Techniques: Fluorescence, Staining, Expressing, RNA Expression, Activation Assay, Sequencing, cDNA Library Assay, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: Isoform switch of CD47 provokes macrophage-mediated pyroptosis in ovarian cancer

doi: 10.1101/2025.04.17.649282

Figure Lengend Snippet: (A) Schematic representation indicated the structure of fusion proteins incorporating GFP or mCherry, CD47 transmembrane domain (TMD) and C-terminal variable regions. (B) Three-dimensional reconstruction confocal image of OVCAR3 cells expressing GFP fused with CD47-L fragment and mCherry fused with CD47-S. (C) Immunofluorescence assay indicated the subcellular location of CD47-S, endoplasmic reticulum (Calnexin), mitochondria (COX IV), and Golgi apparatus (GM130) in OVCAR3. (D-E) Mean florescence intensity (MFI) of surface and intracellular CD47 in OVCAR3 (D) and HEY (E) cells detected by cell flowmetry. Unstained cells are shown in grey. (F) Immunofluorescence assay detecting surface and cytoplasmic CD47 expression in OVCAR3 and HEY cells. OVCAR3 was representative CD47-L dominant (L/S high) cell line while HEY was CD47-S dominant (L/S low). (G) Plasma and organelle membrane protein isolation assay detecting GFP-CD47-L and GFP-CD47-S subcellular location. C, cytosol; PM, plasma membrane; OM, organelle membrane. (H) MFI of membrane CD47 of HEY cells overexpressing CD47-L and CD47-S detected by cell flowmetry. (I) Phagocytotic cell percentage of HEY cells overexpressing CD47-L and CD47-S. Dil and Dio double positive cell percentage of HEY overexpressing CD47-L and CD47-S co-cultured with macrophages. (J) Luciferase signals of subcutaneous injected immunodeficient mouse and photon flux quantification. Immunodeficient mouse were injected with luciferase-expressing HEY cells with CD47-L or CD47-S overexpression (n = 5). THP1-derived macrophages were replenished through the tail vein every three days from the onset of stable tumor formation. (K-L) Total flux (K) and tumor weight (L) of the xerograph mouse model after CD47-L/S overexpression. (M) TUNEL assay detecting dead cells in CD47-L and CD47-S overexpressing xerograph tumor. (N) Affinity assay of SIRPα protein with OVCAR3-GFP cells overexpressing CD47-L and CD47-S. The recombined SIRPα protein was immobilized at the bottom of the container and the fluorescence intensity was detected after gentle washing.

Article Snippet: GFP-CD47-L, a plasmid coding GFP tagged CD47 with its own short 3’UTR, were purchased from Addgene (GFP_CD47_SU, #65473).

Techniques: Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Isolation, Cell Culture, Luciferase, Injection, Over Expression, Derivative Assay, TUNEL Assay, Fluorescence, Gentle

Journal: bioRxiv

Article Title: Isoform switch of CD47 provokes macrophage-mediated pyroptosis in ovarian cancer

doi: 10.1101/2025.04.17.649282

Figure Lengend Snippet: (A) Minimum distortion embedding of single cell CRISPR data where each dot represents a genetic perturbation. Core splicing factor which induced CD47-L (Blue) and CD47-S (Red) switch tendency are annotated. (B) Distribution of medium of CD47 SpliZ value. SpliZ values of cells with core splicing factors perturbation were compared with non-targeted cells. Perturbation induced CD47-L and CD47-S switch were colored in blue and red. (C) Violin plot of the cells belonging to each core splicing factor perturbation, comparing to the non-targeted cells. (D) RNA pull-down coupled with mass spectrometry screening for CD47 intron interacting RNA binding proteins (RBPs) and their belonging spliceosome complex. (E) Venn diagram of the 5 common intron interacting RBPs whose expression was both correlated with CD47 multi-exon skipping PSI and isoform percentage. (F) Correlation analysis between CD47 micro-exons skipping PSI and HNRNPA1 mRNA expression. (G) mRNA expression levels of HNRNPA1, CD47 and its isoforms after knockdown HNRNPA1 in HEY cells. (H) Genomic visualization around CD47 exon 9 and 10 of MicroAS-seq data after HNRNPA1 knockdown in OVCAR8, OVCAR3, and HEY cells. (I) Representative images and relative fluorescence intensity quantification of CD47 multi-exon skipping reporter system after HNRNPA1 overexpression and knockdown in HEY cells. (J) HNRNPA1 crosslink signal density detected by LACE-seq2, eCLIP-seq, and CLIP-seq related to the binding peaks identified by LACE-seq2. (K) Venn diagram of the binding genes identified by LACE-seq2, eCLIP-seq, and CLIP-seq. (L) Genomic visualization of CLIP-seq and LACE-seq binding signals of HNRNPA1, HNRNPH1, HNRNPA2B1, HNRNPF, HNRNPM. HNRNPA1 and HNRNPH1 binding sites predicted by RBPmap based on known motifs. Y-axis: 0-10. (M) RNA pull-down assay indicated HNRNPA1 and HNRNPH1 interacts with CD47 intron fragment 1 and 2. HuR, which was detected to interact with AR-3’UTR, was the positive control while splicing factor was the negative control. (N) Modified dot blot assays were performed with RNA fragments 1 and 2 immobilized onto nylon membranes and their interaction with gradient concentrations of HNRNPA1 was determined. (O) RNA EMSA assay detecting the interaction of HNRNPA1 with CD47 intron fragment 1 and 2.

Article Snippet: GFP-CD47-L, a plasmid coding GFP tagged CD47 with its own short 3’UTR, were purchased from Addgene (GFP_CD47_SU, #65473).

Techniques: CRISPR, Mass Spectrometry, RNA Binding Assay, Expressing, Knockdown, Fluorescence, Over Expression, Binding Assay, Pull Down Assay, Positive Control, Negative Control, Modification, Dot Blot

Journal: bioRxiv

Article Title: Isoform switch of CD47 provokes macrophage-mediated pyroptosis in ovarian cancer

doi: 10.1101/2025.04.17.649282

Figure Lengend Snippet: (A) Schematic map of the location on the genome of ASOs targeting CD47 multiple exons skipping. (B) Normalized exon inclusion level detected by MicroAS-seq after ASO screening in OVCAR3 cells (n=3 biological replicates). (C) CD47-L and total CD47 protein expression after ASOs transfection. The bands were quantified and normalized to NC group. (D) CD47-L and total CD47 protein expression after transfection with gradient concentrations of ASO-I10. (E) Relative CD47-L/CD47-S ratio detected by isoform specific qPCR after 50 nM and 100 nM ASO-I10 transfection. (F) Half maximum effective ASO-I10 concentration for inducing CD47-L to CD47-S conversion. (G) Normalized fluorescence intensity of OVCAR3-L/S-reporter cell line after ASO-NC, ASO-I10 transfection, detected by long-term imaging of living cells. (H) Mean florescence intensity of surface and intracellular CD47 in OVCAR3 cells after ASO-I10 transfection. (I) Phagocytosis ratio after ASO-NC, ASO-I9b, and ASO-I10 transfection in OVCAR3 and HEY cells. (J) Representative fluorescence images of macrophages (Red) co-cultured with OVCAR3 (Green) superimposed on light microscopy images (Up). The live and dead cells of the co-culture system were also stained with PI (Red) and Calcein-AM (Green) (Down). (K) OVCAR3 cells 3D spheroids (n = 6 per group) were co-cultured with macrophages and treated using ASO-I10. Normalized GFP fluorescence intensities were quantified.

Article Snippet: GFP-CD47-L, a plasmid coding GFP tagged CD47 with its own short 3’UTR, were purchased from Addgene (GFP_CD47_SU, #65473).

Techniques: Expressing, Transfection, Concentration Assay, Fluorescence, Imaging, Cell Culture, Light Microscopy, Co-Culture Assay, Staining

Journal: bioRxiv

Article Title: Isoform switch of CD47 provokes macrophage-mediated pyroptosis in ovarian cancer

doi: 10.1101/2025.04.17.649282

Figure Lengend Snippet: (A) Time-lapse photographs of ASO-NC and ASO-I10-transfected OVCAR3-GFP in co-culture with Dil-stained macrophages within 100 mins. (B) Time-lapse photographs of HEY-GFP (CD47-L/S low) in co-culture with Dil-stained macrophages within 120 mins. (C-D) Representative photographs of HEY or ASO-transfected OVCAR3 cancer cells co-cultured with macrophages captured by light microscopy (C) and transmission electron microscopy (D) . (E) Representative images of scanning electron microscopy of ASO-I10 transfected OVCAR3 and OVCAR8 cells after co-culture with macrophages. (F) Distribution of PAX8 (Green) and Cleaved-GSDMD (Red) in ASO-transfected OVCAR3 cells co-cultured with macrophages was determined using immunofluorescence. (G) Protein expression of pyroptosis markers after ASO transfected OVCAR3 cells co-cultured with macrophages. (H, I) IL18 and IL1beta concentration in supernatants of ASO-transfected OVCAR3 cells co-cultured with macrophages. (J) CD47 SpliZ index in scRNA-seq data from a macrophage and cancer cell co-culture system following ASO-I10 treatment, as detected by targeted amplification using scMicroAS-seq. (K) UMAP projection of cell clusters in scRNA-seq of macrophage co-cultured with ASO-I10 transfected OVCAR3 cells. (L) Proportions of epithelial cancer cells and macrophages clusters with ASO-NC and ASO-I10 transfection. (M) Pseudotime trajectory of the macrophage cluster co-cultured with ASO-NC and ASO-I10 transfected OVCAR3 cells, colored by macrophage clusters. (N) Pseudotime trajectory of the macrophage cluster within the co-culture system. The enriched pathways of marker genes of each stage were annotated. (O) Luciferase signals of subcutaneous injected immunodeficient mouse and photon flux quantification. Immunodeficient mouse were injected with luciferase-expressing HOC7 cells mixed with primary human fibroblast (n = 6). THP1-derived macrophages were supplemented through the tail vein after stable tumor formation. LNP packaged ASO was delivery through intratumor injection from Day13. (P-Q) Tumor volume and weight of the xerograph mouse model after ASO treatment and macrophage supplement starting from Day 13. (R) Immunofluorescence and TUNEL images of xenograft tumor model of ASO-NC and ASO-I10 groups. PANCK (green) represents epithelial tumor cells and CD68 (red) represents macrophages.

Article Snippet: GFP-CD47-L, a plasmid coding GFP tagged CD47 with its own short 3’UTR, were purchased from Addgene (GFP_CD47_SU, #65473).

Techniques: Transfection, Co-Culture Assay, Staining, Cell Culture, Light Microscopy, Transmission Assay, Electron Microscopy, Immunofluorescence, Expressing, Concentration Assay, Amplification, Marker, Luciferase, Injection, Derivative Assay, TUNEL Assay