mouse cd47 ecd fc  (Sino Biological)

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: Cell Reports Medicine

Article Title: Cancer vaccine from intracellularly gelated tumor cells functionalized with CD47 blockage and damage-associated molecular pattern exposure

doi: 10.1016/j.xcrm.2025.102092

Figure Lengend Snippet: DAMP exposure and CD47 blockage strengthened the immunogenicity of CD47-DOX-GMC through induction of “eat me signal” and inhibition of “don’t eat me signal” (A) Diagram on the construction of CD47-DOX-GMC. (B) Western blot analysis on the CRT and CD47 expression of GMC and CD47-DOX-GMC. (C) Confocal fluorescence imaging on the green fluorescence probe-labeled CD47 protein and red fluorescence probe-labeled CRT protein of GMC and CD47-DOX-GMC. (D) Schematic illustration on the improved recognition of macrophage and DC toward CD47-DOX-GMC through induction of CD91-CRT interaction and blockage of SIRPα-CD47 interaction. (E) Confocal fluorescence imaging on the phagocytosis behavior of macrophage toward GMC and CD47-DOX-GMC after incubation for 6 h. Red: DiI-stained macrophage. Green: CFSE-stained GMC and CD47-DOX-GMC. (F) Confocal fluorescence imaging on the phagocytosis behavior of DC toward GMC and CD47-DOX-GMC after incubation for 6 h. Red: DiI-stained DC. Green: CFSE-stained GMC and CD47-DOX-GMC. (G and H) Quantification on the phagocytosis rate of macrophage (G) and DC (H) by flow cytometry. (I) The ratio of F4/80 + CD86 + cell in macrophage after treatment with GMC and CD47-DOX-GMC for 6 h. (J) The levels of TNF-α and IL-1β released from macrophage treated with GMC and CD47-DOX-GMC for 6 h. (K) The number and relative abundance of upregulated immune response-related proteins in macrophages treated with GMC and CD47-DOX-GMC, respectively. (L) Confocal fluorescence imaging on the internalization and intracellular location of CD47-DOX-GMC in DC following 1 h incubation. (M) The ratio of CD80 + CD11c + cell in DC after treatment with GMC and CD47-DOX-GMC for 6 h. (N) The number and relative abundance of upregulated immune response-related proteins in DC treated with GMC and CD47-DOX-GMC, respectively. The experiments were repeated three times ( n = 3), and data were presented as mean ± SD. Statistical analyses for (B), (K), and (N) were performed using two-way ANOVA. All other analyses were conducted using one-way ANOVA. ∗∗ p ≤ 0.01 and ∗∗∗ p ≤ 0.001.

Article Snippet: anti-mouse CD47 , Proteintech , Cat#20305-1-AP; RRID: AB_10732838.

Techniques: Immunopeptidomics, Inhibition, Western Blot, Expressing, Fluorescence, Imaging, Labeling, Incubation, Staining, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Cancer vaccine from intracellularly gelated tumor cells functionalized with CD47 blockage and damage-associated molecular pattern exposure

doi: 10.1016/j.xcrm.2025.102092

Figure Lengend Snippet: CD47-DOX-GMC effectively prevented tumor and treated pre-existing tumor (A) 6-week-old C57BL/6 male mice were i.p. inoculated with PBS and CD47-DOX-GMC at a dose of 10 6 per mouse ( n = 6), and then challenged with B16-F10-Luc cells. (B) Tumor bioluminescence was monitored by using IVIS at different time points (day 0, 3, 7, 11, 15, and 19). (C and D) The recorded survival rate curve (C) and semi-quantification on the change of bioluminescence intensity in melanoma-bearing mice (D). (E) 6-week-old C57BL/6 male mice were i.p. injected with B16-F10-Luc cells for 3 days, and then inoculated with PBS, 10 mg/kg of hydrogel, GMC, DOX-GMC, and CD47-DOX-GMC at a dose of 10 6 per mouse ( n = 5), with two doses in total at day 0 and day 7. (F) Tumor bioluminescence was monitored by using IVIS at different time points (day 0, 4, 8, 13, and 19). (G and H) The recorded survival rate curve (G) and semi-quantification on the bioluminescence intensity in melanoma-bearing mice (H). The data in (D) and (H) were presented as mean ± SD.

Article Snippet: anti-mouse CD47 , Proteintech , Cat#20305-1-AP; RRID: AB_10732838.

Techniques: Injection

Journal: Cell Reports Medicine

Article Title: Cancer vaccine from intracellularly gelated tumor cells functionalized with CD47 blockage and damage-associated molecular pattern exposure

doi: 10.1016/j.xcrm.2025.102092

Figure Lengend Snippet: CD47-DOX-GMC significantly activated tumor-associated lymphocytes and cytokines to achieve antitumor therapeutic effect (A and B) 6-week-old C57BL/6 male mice were i.p. injected with B16-F10-Luc cells for 3 days, and then inoculated with PBS, 10 mg/kg of hydrogel, GMC, DOX-GMC, and CD47-DOX-GMC at a dose of 10 6 per mouse ( n = 3), with two doses in total at day 0 and day 7. The peritoneal fluid and spleen were collected at day 12, and T cell type and activation status were analyzed by flow cytometry. Percent of CD4 + and CD8 + T cells with effector memory (CD44 + CD62L low ) phenotypes in ascites (A) and spleen (B) from treated mice. (C) Percent of CD4 + cells expressing activation marker IFN-γ, regulatory T cell markers FoxP3 and CTLA4, and TH1 surface markers and transcription markers in ascites from treated mice. (D) Percent of CD8 + cells expressing activation markers TNF-α and IFN-γ and exhaustion markers PD-1 and granzyme B in ascites from treated mice. (E and F) Cytokine analysis on ascites TNF-α (E) and IFN-γ (F) from treated mice. (G) Tumor cell death in co-cultures containing peritoneal CD8 + T cells from naive or vaccinated mice. (H) Tumor-naive 6-week-old C57BL/6 male mice were i.p. injected with peritoneal 2 × 10 5 magnetically enriched CD8 + cell, and then challenged with B16-F10-Luc cells after 1 day ( n = 5). (I) Tumor bioluminescence was monitored by using IVIS at different time points (day 0, 4, 8, 13, and 19). (J) Semi-quantification on the change of bioluminescence intensity in melanoma-bearing mice. The experiments except in vivo antitumor imaging were repeated three times ( n = 3), and data were presented as mean ± SD. All statistical analyses were conducted using one-way ANOVA. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.

Article Snippet: anti-mouse CD47 , Proteintech , Cat#20305-1-AP; RRID: AB_10732838.

Techniques: Injection, Activation Assay, Flow Cytometry, Expressing, Marker, In Vivo, Imaging

Journal: Cell Reports Medicine

Article Title: Cancer vaccine from intracellularly gelated tumor cells functionalized with CD47 blockage and damage-associated molecular pattern exposure

doi: 10.1016/j.xcrm.2025.102092

Figure Lengend Snippet: Immune checkpoint inhibitor facilitated the T cell-mediated tumor-killing effect induced by CD47-DOX-GMC on established tumor (A) 6-week-old C57BL/6 male mice were i.p. inoculated with B16-F10-Luc cells for 8 days to establish melanoma-bearing mice and i.p. treated with GMC, DOX-GMC, and CD47-DOX-GMC at a dose of 10 6 per mouse for two doses in total at day 0 and day 7 ( n = 5). (B) Tumor bioluminescence was monitored by using IVIS at different time points (day 0, 4, and 8). (C and D) The recorded survival rate curve (C) and semi-quantification on the change of bioluminescence intensity in melanoma-bearing mice (D). (E) Melanoma mice were additionally i.p. administered with aPD-L1 after CD47-DOX-GMC treatment for 3 days ( n = 5), with a dose of 4 mg/kg at day 3 and day 10, respectively. (F) Tumor bioluminescence was monitored by using IVIS at different time points (day 0, 4, 8, 13, and 19). (G and H) The recorded survival rate curve (G) and semi-quantification on the change of bioluminescence intensity in melanoma-bearing mice (H). The data in (D) and (H) were presented as mean ± SD.

Article Snippet: anti-mouse CD47 , Proteintech , Cat#20305-1-AP; RRID: AB_10732838.

Techniques:

Journal: Cell Reports Medicine

Article Title: Cancer vaccine from intracellularly gelated tumor cells functionalized with CD47 blockage and damage-associated molecular pattern exposure

doi: 10.1016/j.xcrm.2025.102092

Figure Lengend Snippet: Personalized CD47-DOX-GMC vaccine from ascites achieved effective antitumor efficacy (A) B16-F10 cells were collected from ascites of tumor-bearing mice and enriched by using filtration capture. (B) 6-week-old C57BL/6 male mice were i.p. administered with B16-F10-Luc cells for 3 days, and then inoculated with ascites-derived CD47-DOX-GMC at a dose of 10 6 per mouse ( n = 5), with two doses in total at day 0 and day 7. (C) Tumor bioluminescence was monitored by using IVIS at different time points (day 0, 4, 8, 13, and 19). (D and E) The recorded survival rate curve (D) and semi-quantification on the change of bioluminescence intensity in melanoma-bearing mice (E). The data in (E) were presented as mean ± SD.

Article Snippet: anti-mouse CD47 , Proteintech , Cat#20305-1-AP; RRID: AB_10732838.

Techniques: Filtration, Derivative Assay

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a Overview of the outline for establishing H1975 and HEK293T cell lines expressing a fusion GFP gene in the C-terminus of CD47 (CD47-GFP) and subsequently identifying the deubiquitinase of CD47 through DUB siRNA library screening with two stable cell lines. DUBs deubiquitinases; FACS fluorescence-activated cell sorting; MFI mean fluorescence intensity. b Whole-cell lysates (WCL) of H1975 or PC9 cells treated with indicated deubiquitinase inhibitors (2.5 µM) or dimethyl sulfoxide (DMSO) for 12 hours (h) were prepared and subjected to immunoblotting (IB) analysis. c – e IB analysis of WCL derived from H1975 cells ( c ) and PC9 cells ( e ) treated with ML364 (1 µM and 2 µM) or DMSO for 16 h. The mRNA level of CD47 in H1975 cells ( c ) was measured using reverse transcription quantitative PCR (RT-qPCR) ( d ). f , g Immunofluorescence (IF) staining for CD47 in H1975 cells ( f ) and PC9 cells ( g ) treated with ML364 (2 µM) for 16 h. Scale bar, 25 μm. h – k IB analysis of WCL derived from H1975 ( h ) or PC9 ( j ) cells stably expressing sh USP2 or sh GFP , respectively. The mRNA level of CD47 in H1975 ( i ) or PC9 ( k ) cells was measured using RT-qPCR. l , m IB analysis of Cd47 protein expression in the tissues of lung, heart, or kidney obtained from wild-type (WT) and Usp2 −/− mice ( l ). Quantification of Cd47 protein band intensity was normalized to vinculin ( m ). n – p IB analysis of WCL derived from HEK293T cells co-transfected with indicated constructs ( n , p ). The mRNA level of CD47 was quantified by RT-qPCR ( o ). EV: empty vector. q , r IB analysis of WCL derived from HEK293T cells co-transfected with indicated constructs. Cells were treated with 200 μg/ml CHX for the indicated time points ( q ). Quantification of CD47 protein band intensity was normalized to vinculin, then compared to the t = 0 time point ( r ). s , t Representative images from immunohistochemical (IHC) staining of CD47 and USP2 in human lung adenocarcinoma ( s ). Scale bar, left panels: 100 μm; right panels: 50 μm. n = 83. Quantification of USP2 and CD47 staining intensities was performed as average optical density (AOD) [AOD = Integrated Optical Density (IOD) SUM/Area SUM] (t) . For ( d , i , k , m , o , and r ), unpaired two-tailed Student’s t -test. Correlations were analyzed by Pearson’s test ( t ). Data are shown as the mean ± SD, n = 3 independent biological replicates. P < 0.05 was considered statistically significant. n = 3 biologically independent experiments for ( b , c , e , h , j , l , n , p , and q ). Source data are provided as the Source Data file.

Article Snippet: InVivoMAb anti-mouse CD47 (IAP) was purchased from BioXCell (BE0270).

Techniques: Expressing, Library Screening, Stable Transfection, Fluorescence, FACS, Western Blot, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining, Transfection, Construct, Plasmid Preparation, Immunohistochemical staining, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a IB analysis of HEK293T WCL and anti-HA immunoprecipitates (IPs). HEK293T cells were co-transfected with indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. b IB analysis of glutathione S-transferase (GST) pull-down protein mixture from HEK293T cell lysates that overexpressed CD47-cHA incubated with bacterially purified recombinant GST or GST-USP2 protein. c IB analysis of GST pull-down products from HEK293T cell lysates that overexpressed Flag-USP2 incubated with bacterially purified recombinant GST or GST-CD47 protein. d Schematic representation of WT and truncations of USP2, including the N-terminal region of amino acid (aa) 1–266 and C-terminal domain of aa267–605. e IB analysis of WCL and anti-HA IPs obtained from HEK293T cells, which were co-transfected with indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. f IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of CD47-cHA incubated with bacterially purified recombinant GST, GST-USP2 WT, and GST-USP2 truncations. g IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of Flag-USP2 WT and truncations incubated with bacterially purified recombinant GST-CD47 protein. h Schematic diagram of CD47 WT and its various deletion mutants. i IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of CD47-cHA WT and deletion mutants incubated with bacterially purified recombinant GST-USP2 protein. j , k IB analysis of WCL and IPs derived from H1975 ( j ) and PC9 ( k ) cells. l , m IB analysis of WCL and Ni-NTA pull-down products of the in vivo ubiquitination assay in the guanidine-HCl denaturing buffer. HEK293T cells were co-transfected with the indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. n IB analysis of WCL and Ni-NTA pull-down products of the in vivo ubiquitination assay in the guanidine-HCl denaturing buffer. HEK293T cells were co-transfected with the indicated constructs and treated with 2 µM ML364 for 16 h and 10 µM MG132 for 12 h before harvesting. o , p IB analysis of WCL and IPs derived from lysates of H1975 ( o ) and PC9 ( p ) cells using indicated K48-Ubi antibodies. Cells were treated with 20 µM MG132 for 6 h before harvesting. q , r IB analysis of WCL and anti-CD47 IPs derived from H1975 ( q ) or PC9 ( r ) cells stably expressing sh USP2 or sh GFP , respectively. Cells were treated with 20 µM MG132 for 6 h before harvesting. n = 3 biologically independent experiments for ( a , b , c , e , f , g , and i – r ). Source data are provided as the Source Data file.

Article Snippet: InVivoMAb anti-mouse CD47 (IAP) was purchased from BioXCell (BE0270).

Techniques: Transfection, Construct, Incubation, Purification, Recombinant, Derivative Assay, Expressing, In Vivo, Ubiquitin Proteomics, Stable Transfection

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a A schematic treatment plan for immunocompetent C57BL/6J mice bearing LLC tumors. Mice were subcutaneously implanted with 2 × 10 6 LLC cells and treated with a control vehicle, USP2 inhibitor (ML364, 5 mg/kg, daily for 12 doses), anti-PD-1 mAb (100 μg per mouse, every three days for 4 treatments), or combined treatment, respectively. i.p., intraperitoneal; s.c., subcutaneous; MAb, monoclonal antibody. b , c Tumor growth ( b ) or Kaplan–Meier survival curves ( c ) for C57BL/6J bearing LLC tumors with indicated treatments. d The weight of LLC tumors was measured at the endpoint. e Quantification of cellular surface CD47 on CD45 − cells in subcutaneous LLC tumors derived from C57BL/6J mice with indicated treatments. f – h Quantification of MHCII + (M1 macrophage) cells ( f ) or CD206 + (M2 macrophage) cells ( g ) represented as percentage of F4/80 + macrophages, and CD8 + T cells ( h ) represented as percentage of CD3 + T cells in subcutaneous LLC tumors derived from C57BL/6J mice-indicated treatments. i A schematic treatment plan for the immunocompetent C57BL/6J lung tumor mouse model. Mice were injected with 1 × 10 6 LLC cells/per mouse via tail vein injection, followed by treatments with control vehicle, ML364 (5 mg/kg), anti-PD-1 mAb (100 μg per mouse), or combined treatment, respectively. i.p., intraperitoneal; i.v., intravenous. j – l Representative H&E-stained images of lung tissues from mice across different groups ( j ), tumor size was quantified by measuring the cross-sectional area of all tumors ( k ), and the tumor-occupied lung area (tumor area, %) was calculated ( l ) from three nonconsecutive sections per mouse. Scale bars, 5 mm. m LLC-GFP tumors bearing C57BL/6J mice were treated as ( a ). Representative images of IF staining for tumor cells (GFP, green), macrophages (F4/80, red), and nuclei (DAPI, blue). The yellow (indicated with arrows) showing colocalization of GFP (green) and F4/80 (red) double-positive cells suggested the macrophage phagocytosis of tumor cells. Each point represents the average counts of yellow cells within three high-power fields. The sections were scanned at 60× with an oil objective on a confocal microscope. Scale bars, 50 μm (left panels); zoom scale bars, 20 μm (right panels). For ( d – h , k , and l ), unpaired two-tailed Student’s t -test. Two-way ANOVA for ( b ). Log-rank test for ( c ). n = 5 mice per group. Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: InVivoMAb anti-mouse CD47 (IAP) was purchased from BioXCell (BE0270).

Techniques: Control, Derivative Assay, Injection, Staining, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a Diagrammatic treatment plan for immunocompetent C57BL/6J mice with LLC tumors. Mice were implanted with 2 × 10 6 LLC cells and treated with Vehicle, ML364 (5 mg/kg daily for 18 doses), anti-PD-1 mAb (100 μg/mouse every three days for 6 doses), or combination therapy. i.p., intraperitoneal; s.c., subcutaneous. b Tumor volume in different treatment groups was assessed using calipers, and the tumor growth curve was generated. n = 8 (Vehicle); 8 (ML364); 8 (anti-PD-1 mAb) and 10 (Combined therapy) mice. c Kaplan–Meier survival curves for C57BL/6J bearing LLC tumors with indicated treatments. Log-rank test. n = 8 (Vehicle); 8 (ML364); 8 (anti-PD-1 mAb) and 10 (Combined therapy) mice. d A schematic treatment plan for immunocompetent C57BL/6J mice bearing 2 × 10 6 LLC tumors. Mice were treated with a control vehicle, USP2 inhibitor (ML364, 30 mg/kg for 18 doses), anti-PD-1 mAb (200 μg per mouse for 6 doses), or combined treatment, respectively. e Tumor volume in the different treatment groups was measured using calipers, and the corresponding tumor growth curve was generated. Vehicle (n = 9), ML364 (n = 8), PD-1 mAb (n = 8), ML364 & PD-1 mAb (n = 13) mice per group. f Kaplan–Meier survival curves for C57BL/6J bearing LLC tumors with indicated treatments. Vehicle (n = 9), ML364 (n = 8), PD-1 mAb (n = 8), ML364 & PD-1 mAb (n = 13) mice per group. Log-rank test. g IHC staining for CD47 and PD-L1 in LLC tumors from C57BL/6J mice treated with indicated reagents. Scale bar, 50 μm. h , i Quantification for CD47 ( h ) and PD-L1 ( i ) positive cells. Each point represents the average counts of positive cells within three high-power fields. For ( h ) and ( i ), unpaired two-tailed Student’s t -test. n = 5 mice per group. Log-rank test for ( c , f ). Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: InVivoMAb anti-mouse CD47 (IAP) was purchased from BioXCell (BE0270).

Techniques: Generated, Control, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a A schematic treatment plan for immunocompetent C57BL/6J mice bearing sg Control- or sg Cd47- LLC tumors. Mice were subcutaneously implanted with 2 × 10 6 sg Control or sg Cd47 -LLC cells and treated with a control vehicle, USP2 inhibitor (ML364, 5 mg/kg, daily for 12 doses), anti-PD-1 mAb (100 μg per mouse, every three days for 4 treatments), or combined treatment, respectively. i.p., intraperitoneal; s.c., subcutaneous. b , c Tumor volume in each treatment group was measured using calipers, and the tumor growth curve was subsequently plotted ( b ). At the study endpoint, the weight of LLC tumors was recorded ( c ). n = 5 mice per group. d The body weight of C57BL/6J mice was monitored throughout the duration of the indicated treatment. n = 5 mice per group. e A schematic treatment plan for CD8 T cell or macrophage deletion experiments. Mice were randomly divided into five groups: (1) vehicle, (2) ML364 & PD-1 mAb (MP), (3) MP & CD8 deletion (αCD8), (4) MP & macrophage deletion (clodronate liposomes, CL), and (5) MP & αCD8 & CL. On day 5 post-tumor implantation, mice in MP were treated with a combination of ML364 (5 mg/kg) daily for 12 treatments and anti-PD-1 mAb (100 μg per mouse) every 3 days for 4 doses. After grouping as indicated, mice received αCD8 antibody (200 μg per mouse) every 3 days for 4 doses, starting three days before tumor implantation. Macrophage deletion by CL was administered with 200 μl per mouse every 3 days for 4 doses, starting on the day of tumor implantation. i.p., intraperitoneal; s.c., subcutaneous. f , g Tumor volume for each treatment group was measured using calipers, and the tumor growth curve was plotted ( f ). The weight of LLC tumors was measured at the endpoint ( g ). n = 5 mice per group. h , i Quantification of CD8 + represented as percentage of CD3 + T cells ( h ) and F4/80 + macrophages represented as percentage of CD11b + cells ( i ) in subcutaneous LLC tumors derived from C57BL/6J mice treated with indicated reagents. For ( c ) and ( g – i ), unpaired two-tailed Student’s t -test. n = 5 mice per group. Two-way ANOVA for ( b , d , and f ). Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: InVivoMAb anti-mouse CD47 (IAP) was purchased from BioXCell (BE0270).

Techniques: Control, Liposomes, Tumor Implantation, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a , b Representative IF staining images for CD163 (green) and CD8 (red) in human lung adenocarcinoma (LUAD) tissues with the USP2 high (n = 13) and low (n = 16) expression ( a ). Quantifications of CD163 and CD8 positive cells in LUAD samples with USP2 high or low expression, respectively ( b ). Each point represents the average counts of positive cells within three high-power fields. Scale bars, 500 μm (left panels); zoom scale bars, 50 μm (right panels). c – e USP2 expression was negatively correlated with the infiltration of M1 macrophages ( c ) and CD8 + T cells ( e ), while USP2 displayed a positive correlation with the infiltration of M2 macrophages ( d ) in lung cancer patients based on the immune association analysis by TIMER2.0 software. f , g Representative images from IHC staining of CD47 and USP2 in responders (R) and non-responders (NR) of lung cancer patients with anti-PD-1 therapy ( f ). Scale bar, left panels: 100 μm; right panels: 50 μm. Association between USP2 or CD47 expression and response to anti-PD-1 therapy in lung cancer patients ( g ). n = 6 (R), or 6 (NR). Patients were divided into USP2 or CD47 low and high expression groups according to their IHC score mean value ( f ). h , i Representative images of multiplex immunohistochemistry (mIHC) staining for R and NR of lung cancer patients with anti-PD-1 immunotherapy ( h ). Quantifications of CD163 (white), CD8 (red), and CK (green) in each group ( i ). Each point represents the average counts of positive cells within three high-power fields. n = 5 per group. Scale bars, 100 μm (left panels); zoom scale bars, 50 μm (right panels). j , k Representative images from IHC staining of CD47 and USP2 in responders and non-responders of oral cancer patients with anti-PD-1 therapy ( j ). Scale bar, left panels: 100 μm; right panels: 50 μm. Association between USP2 or CD47 expression and response to anti-PD-1 therapy in oral cancer patients ( k ). n = 25 (R), or 19 (NR). Patients were divided into USP2 or CD47 low and high expression groups according to their IHC score mean value ( j ). l , m Representative images of mIHC staining for responders and non-responders of oral cancer patients with anti-PD-1 therapy ( l ). Quantifications of CD163 (white), CD8 (red), and CK (green) in each group ( m ). Each data point represents the average counts of positive cells within three high-power fields. n = 18 (R), or 13 (NR). Scale bars, 100 μm (left panels); zoom scale bars, 50 μm (right panels). For ( b ), unpaired two-tailed Student’s t -test. For ( g ) and ( k ), a two-sided χ 2 test. For ( i ) and ( m ), two-tailed Non-parametric Mann–Whitney test. Data are shown as the mean ± SD P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: InVivoMAb anti-mouse CD47 (IAP) was purchased from BioXCell (BE0270).

Techniques: Staining, Expressing, Software, Immunohistochemistry, Multiplex Assay, Two Tailed Test, MANN-WHITNEY