rabbit anti human integrin αv  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: Cancer Immunotherapy via Disruption of Integrin αvβ3 and CD47 Costabilization on Cancer Cell Surface

doi: 10.1002/advs.202501602

Figure Lengend Snippet: Coexpression of CD47 and integrin αvβ3 on tumor cells and in clinical breast cancer samples. A) A correlation analysis across selected tumor lines—MCF‐7, LoVo, HCT116, A549, MDA‐MB‐231, U87, H23, MDA‐MB‐468, and Hs578T—demonstrated a robust positive relationship between αvβ3 and CD47 expression ( R 2 = 0.830, p < 0.0001). Notably, this correlation did not extend to nontumorigenic cell lines MCF‐10A and LO2, the hematopoietic tumor cell line HL60, or the he triple‐positive (ER + /PR + /HER2 + ) BT474 breast cancer line, which collectively exhibited divergent expression profiles. B,C) MDA‐MB‐231 cells and red blood cells (RBCs) were labeled and analyzed for CD47 and αvβ3 expression by flow cytometry. Pooled data for CD47 and αvβ3 cell surface markers are presented. D) Schematic representation of tissue microarray (TMA) for clinical breast cancer tissue samples along with paired paraneoplastic and normal tissues. E) Representative images of multiplex fluorescent immunohistochemical staining showing DAPI + (blue), αvβ3 + (green), CD47 + (red), CD68 + (sky blue), and CK + (grayish purple) in breast cancer tissue. Enlarged areas within dashed boxes are shown in the right panel. A merged image of all channels is displayed at the bottom. Scale bar = 50 µm. F,G) Statistical analysis of the proportion of αvβ3 and CD47 double‐positive cells in breast cancer and normal/paracarcinoma tissues. All data are expressed as mean ± s.e.m.; *** p < 0.001 versus control, unpaired t ‐test (C, D, G, and H); ns, not significant.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies, secondary antibodies anti‐rabbit IgG HRP‐conjugated (1: 3000, 7074S, Cell Signaling Technology) and anti‐mouse IgG HRP‐conjugated (1: 3000, 7076S, Cell Signaling Technology) at 25 °C for 2 h. The primary antibodies used were GAPDH monoclonal antibody (1: 1,0000, 60004‐1‐Ig, Proteintech), CD47 antibody (1: 500, sc‐12730, Santa Cruz), integrin αv antibody (1: 1000, 4711S, Cell Signaling Technology), integrin β3 antibody (1: 1000, 13166S, Cell Signaling Technology).

Techniques: Expressing, Labeling, Flow Cytometry, Microarray, Multiplex Assay, Immunohistochemical staining, Staining, Control

Journal: Advanced Science

Article Title: Cancer Immunotherapy via Disruption of Integrin αvβ3 and CD47 Costabilization on Cancer Cell Surface

doi: 10.1002/advs.202501602

Figure Lengend Snippet: Decreased surface expression of αvβ3 or CD47 significantly reduces the presence of the other on the cancer cell surface. A) Schematic representation of αvβ3 involvement in the CD47–SIRPα axis on the cell surface. B–E) MDA‐MB‐231 cells were transfected with siRNA targeting integrin subunit αv (B,C) or β3 (D,E) for 48 h. Flow cytometry was then performed, and pooled data for cell surface markers αvβ3 and CD47 were analyzed. F,G) Flow cytometry analysis of MDA‐MB‐231 cells transfected with siRNA targeting CD47 for 48 h, followed by pooled data analysis of cell surface markers αvβ3 and CD47. H–K) MDA‐MB‐231 cells were transfected with lentiviruses targeting integrin subunit αv (H, I) or β3 (J, K) to generate stable cell lines. Flow cytometry was performed, and pooled data for cell surface markers αvβ3 and CD47 were analyzed. All data are expressed as mean ± s.e.m., n = 3 independent experiments; ** p < 0.01, *** p < 0.001 versus control, unpaired ‐test (H–K); one‐way ANOVA with Dunnett's post‐hoc test (B–G); (B), ( F (2, 6) = 264.1, p < 0.0001), (C), ( F (2, 6) = 244.7, p < 0.0001), (D), ( F (2, 6) = 34.96, p = 0.0005, (E), ( F (2, 6) = 25.27, p = 0.0012), (F), ( F (2, 6) = 59.25, p = 0.0001), (G), ( F (2, 6) = 1497, p < 0.0001).

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies, secondary antibodies anti‐rabbit IgG HRP‐conjugated (1: 3000, 7074S, Cell Signaling Technology) and anti‐mouse IgG HRP‐conjugated (1: 3000, 7076S, Cell Signaling Technology) at 25 °C for 2 h. The primary antibodies used were GAPDH monoclonal antibody (1: 1,0000, 60004‐1‐Ig, Proteintech), CD47 antibody (1: 500, sc‐12730, Santa Cruz), integrin αv antibody (1: 1000, 4711S, Cell Signaling Technology), integrin β3 antibody (1: 1000, 13166S, Cell Signaling Technology).

Techniques: Expressing, Transfection, Flow Cytometry, Stable Transfection, Control

Journal: Advanced Science

Article Title: Cancer Immunotherapy via Disruption of Integrin αvβ3 and CD47 Costabilization on Cancer Cell Surface

doi: 10.1002/advs.202501602

Figure Lengend Snippet: Knockdown of integrin αv expression decelerates orthotopic tumor growth in the mammary fat pad of nude mice. A) THP‐1‐derived macrophages were labeled and analyzed by flow cytometry for SIRPα expression over 48 h, with pooled data for the cell surface marker SIRPα. B,C) siRNA‐mediated knockdown of the integrin αv subunit enhances phagocytosis by THP‐1‐derived macrophages on MDA‐MB‐231‐Cherry cells. (B) Representative fluorescence microscopy images of phagocytic activity and (C) quantitative analysis of the mean fluorescence intensity signal in confocal images. Scale bar = 50 µm. D) MDA‐MB‐231 shNC or MDA‐MB‐231 shαv tumor cells (2 × 10 6 ) were implanted in the mammary fat pad of female nude mice. Tumor growth kinetics were compared over 33 days (8 mice per group). E) Representative images of mice transplanted with MDA‐MB‐231 shNC versus MDA‐MB‐231 shαv tumors on day 33 (images represent two independent experimental groups, 8 mice per group). Scale bar = 1 cm. F) Representative images of tumor size and tumor weight analysis of MDA‐MB‐231 shNC and MDA‐MB‐231 shαv tumors. Scale bar = 1 cm. G) Representative flow cytometry plots showing macrophage phagocytosis of Cherry+ MDA‐MB‐231 shNC tumors (left) versus MDA‐MB‐231 shαv tumors (middle). Numbers indicate the frequency of phagocytic events in all macrophage infiltrates. Quantification of all macrophage infiltrates (right) was assessed for MDA‐MB‐231 (Cherry)+/F4/80 (APC)+. Eight experimental replicates are represented. H,I) Flow cytometric analysis of phagocytic activity by bone‐marrow‐derived (H) and primary peritoneal (I) macrophages from NSG mice toward ITGAV‐stably silenced MDA‐MB‐231 cells and control counterparts. All data are expressed as mean ± s.e.m.; * p < 0.05, ** p < 0.01, *** p < 0.001 versus control, unpaired t ‐test (A, C, D, F–I).

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies, secondary antibodies anti‐rabbit IgG HRP‐conjugated (1: 3000, 7074S, Cell Signaling Technology) and anti‐mouse IgG HRP‐conjugated (1: 3000, 7076S, Cell Signaling Technology) at 25 °C for 2 h. The primary antibodies used were GAPDH monoclonal antibody (1: 1,0000, 60004‐1‐Ig, Proteintech), CD47 antibody (1: 500, sc‐12730, Santa Cruz), integrin αv antibody (1: 1000, 4711S, Cell Signaling Technology), integrin β3 antibody (1: 1000, 13166S, Cell Signaling Technology).

Techniques: Knockdown, Expressing, Derivative Assay, Labeling, Flow Cytometry, Marker, Fluorescence, Microscopy, Activity Assay, Stable Transfection, Control

Journal: Advanced Science

Article Title: Cancer Immunotherapy via Disruption of Integrin αvβ3 and CD47 Costabilization on Cancer Cell Surface

doi: 10.1002/advs.202501602

Figure Lengend Snippet: Integrin αvβ3‐specific inhibitors reduce CD47 expression and enhance macrophage‐mediated tumor phagocytosis. A,B) Representative fluorescence microscopy images of MDA‐MB‐231 cell phagocytosis by macrophages (Protonex Red+, red) after 36 h of coculture with either IgG control or integrin αvβ3 activating antibody (23C6, 10 µg mL −1 ) (A), and quantitative analysis of mean fluorescence intensity in confocal images (B). Scale bar: 50 µm. C,D) Flow cytometry analysis of CD47 surface expression in MDA‐MB‐231 cells treated with various concentrations of the integrin αvβ3 inhibitors cilengitide (C) or cyclo(─RGDfK) (0, 0.5, 1, 2, and 4 µ m ) (D) for 48 h, including pooled data. E–H) Representative images from live cell microscopy phagocytosis assays of Protonex Red+ MDA‐MB‐231 cells treated with the integrin αvβ3 inhibitor cyclo(─RGDfK) (E) or cilengitide (4 µ m ) (G) for 36 h, and quantitative analysis of mean fluorescence intensity in confocal images (F, H). Scale bar: 50 µm. I,J) Representative images from live cell microscopy phagocytosis assays of Protonex Red+ MDA‐MB‐231 cells treated with the integrin αv inhibitor CWHM‐12 (4 µ m ) (I), and quantitative analysis of mean fluorescence intensity in confocal images (J). Scale bar: 50 µm. All data are expressed as mean ± s.e.m., n = 3 independent experiments; * p < 0.05, ** p < 0.001, and *** p < 0.001 versus control, unpaired t ‐test (B, F, H and J); one‐way ANOVA with Dunnett's post‐hoc test (C and D); (C), ( F (4, 10) = 162.8, p < 0.0001), (D), ( F (4, 10) = 25.18, p < 0.0001); ns, not significant.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies, secondary antibodies anti‐rabbit IgG HRP‐conjugated (1: 3000, 7074S, Cell Signaling Technology) and anti‐mouse IgG HRP‐conjugated (1: 3000, 7076S, Cell Signaling Technology) at 25 °C for 2 h. The primary antibodies used were GAPDH monoclonal antibody (1: 1,0000, 60004‐1‐Ig, Proteintech), CD47 antibody (1: 500, sc‐12730, Santa Cruz), integrin αv antibody (1: 1000, 4711S, Cell Signaling Technology), integrin β3 antibody (1: 1000, 13166S, Cell Signaling Technology).

Techniques: Expressing, Fluorescence, Microscopy, Control, Flow Cytometry

Journal: Advanced Science

Article Title: Cancer Immunotherapy via Disruption of Integrin αvβ3 and CD47 Costabilization on Cancer Cell Surface

doi: 10.1002/advs.202501602

Figure Lengend Snippet: Blocking αvβ3 activation reduces surface expression of αvβ3 and CD47 on MDA‐MB‐231 cells. A) Schematic representations of the activated and inactive states of integrin αvβ3. B) Flow cytometry analysis of CD47 surface expression on MDA‐MB‐231 cells treated with various concentrations of the integrin αvβ3 inhibitor RGDS (0, 1, 2, and 4 µ m ) for 48 h, including pooled data. C,D) Representative fluorescence microscopy images of in vitro phagocytosis of MDA‐MB‐231 cells by macrophages (Protonex Red+, red) after 36 h of coculture with either IgG control or the integrin αvβ3 nonactivating antibody (LM609, 10 µg mL −1 ) (C), and quantitative analysis of mean fluorescence intensity in confocal images (D). Scale bar: 50 µm. E,F) Flow cytometry analysis of αvβ3 and CD47 surface expression on MDA‐MB‐231 cells treated with fibronectin (FN, 20 µg mL −1 ) (E) or EGTA (2 m m ) (F) for 48 h, with pooled data of surface markers αvβ3 and CD47. G) Flow cytometry analysis of αvβ3 and CD47 surface expression on MDA‐MB‐231 cells after siRNA‐mediated knockdown of Talin‐1 for 48 h, including pooled data of surface markers αvβ3 and CD47. H) Flow cytometry analysis of αvβ3 and CD47 surface expression in MDA‐MB‐231 cells 48 h after siRNA‐mediated knockdown of Kindlin‐2, with pooled data of surface markers αvβ3 and CD47. I) Flow cytometry analysis of αvβ3 and CD47 surface expression in MDA‐MB‐231 cells 48 h after combined siRNA‐mediated knockdown of the αv subunit and Talin‐1, with pooled data for surface markers αvβ3 and CD47. All data are expressed as mean ± s.e.m., n = 3 independent experiments; * p < 0.05, ** p < 0.001, *** p < 0.001 versus control, unpaired t ‐test (D–H); one‐way ANOVA with Dunnett's post‐hoc test (B and I); (B), ( F (3, 8) = 0.4324, p = 0.7356), (I), ( F (3, 8) = 134.3, p < 0.0001), ( F (3, 8) = 71.77, p < 0.0001). ns, not significant.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies, secondary antibodies anti‐rabbit IgG HRP‐conjugated (1: 3000, 7074S, Cell Signaling Technology) and anti‐mouse IgG HRP‐conjugated (1: 3000, 7076S, Cell Signaling Technology) at 25 °C for 2 h. The primary antibodies used were GAPDH monoclonal antibody (1: 1,0000, 60004‐1‐Ig, Proteintech), CD47 antibody (1: 500, sc‐12730, Santa Cruz), integrin αv antibody (1: 1000, 4711S, Cell Signaling Technology), integrin β3 antibody (1: 1000, 13166S, Cell Signaling Technology).

Techniques: Blocking Assay, Activation Assay, Expressing, Flow Cytometry, Fluorescence, Microscopy, In Vitro, Control, Knockdown

Journal: Advanced Science

Article Title: Cancer Immunotherapy via Disruption of Integrin αvβ3 and CD47 Costabilization on Cancer Cell Surface

doi: 10.1002/advs.202501602

Figure Lengend Snippet: Direct interaction between CD47 and integrin αvβ3 in cancer cells. A) MDA‐MB‐231 cell lysates were immunoprecipitated with control IgG antibody and anti‐CD47 antibody. Co‐immunoprecipitation (Co‐IP) revealed the presence of ITGαv and ITGβ3 in proteins precipitated by the CD47 antibody, indicating a direct interaction between CD47 and integrin αvβ3 in CD47‐expressing MDA‐MB‐231 cells. B) Optimized model showing CD47 (yellow) interacting with integrin αvβ3 (red and blue) on the same cell, or with SIRPα (grey) on macrophages through its IgV domain. C) Schematic diagram of the extracellular region sequence of CD47. D) Optimized model of CD47 interaction with αvβ3 obtained using Z‐dock and conformation‐enhanced sampling. This model identified the T51–Y65 region as a key site in CD47 for interaction with αvβ3, leading to the design of an interfering peptide (green, PSFL‐NK13 M46‐K57 ). E,F) Lysates of HEK293 cells expressing Flag–ITGαv or Myc–ITGβ3 and HA–CD47 (HA–CD47: K26–F32, T44–N50, T51–Y55, G62–T67, D69–N73, D80–K85) were analyzed. HA signal was detected in proteins precipitated with Flag or Myc antibodies. Co‐IP signal for Myc was reduced in cells coexpressing Myc–ITGβ3 and HA–CD47 Δ51–55 . These results were consistently observed in three independent experiments. G) Schematic diagram illustrating the detection of the CD47, ITGαv, and ITGβ3 ternary complex using BRET (Bioluminescence Resonance Energy Transfer). H) Regression curves presenting the net BRET signals in HEK293T cells transfected with CD47–Rluc8, ITGαv–eYFP, and ITGβ3–eYFP, n = 3–7; * p < 0.05, *** p < 0.001, **** p < 0.0001 when compared to BRET signals without ITGαv–eYFP or ITGβ3–eYFP, F (10, 66) = 6.538, Two‐way ANOVA. I) Evaluation of the impact of PSFL‐NK13 (20 µ m ) on the BRET signal corresponding to the CD47–Rluc8, ITGαv–eYFP, and ITGβ3–eYFP ternary complex. n = 5–6; **** p < 0.0001 compared to vehicle, unpaired t ‐test. All data are presented as mean ± s.e.m.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies, secondary antibodies anti‐rabbit IgG HRP‐conjugated (1: 3000, 7074S, Cell Signaling Technology) and anti‐mouse IgG HRP‐conjugated (1: 3000, 7076S, Cell Signaling Technology) at 25 °C for 2 h. The primary antibodies used were GAPDH monoclonal antibody (1: 1,0000, 60004‐1‐Ig, Proteintech), CD47 antibody (1: 500, sc‐12730, Santa Cruz), integrin αv antibody (1: 1000, 4711S, Cell Signaling Technology), integrin β3 antibody (1: 1000, 13166S, Cell Signaling Technology).

Techniques: Immunoprecipitation, Control, Co-Immunoprecipitation Assay, Expressing, Sequencing, Sampling, Bioluminescence Resonance Energy Transfer, Transfection