anti human asc antibody  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Targeting Siglec-10/α3β1 Integrin Interactions Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1101/2025.05.06.652455

Figure Lengend Snippet: (a) Experimental design for the pull-down of Siglec-10 ligands on PDAC cells. Recombinant Siglec-10 (as well as no-protein control or Siglec-5 control) Fc was allowed to bind its physiological ligands on the surface of PDAC cells, followed by an HRP-conjugated anti-Fc secondary antibody. In the presence of H O, HRP generated short-lived radicals that facilitated the transfer of biotin to proximal Siglec-10 ligands. Biotinylated Siglec-10 ligands were pulled down using streptavidin beads and identified by mass spectrometry. (b) A total of 4,044 proteins were identified, with enriched binding compared to a control using the anti-Fc antibody only without Siglec-10 protein. Of these, 110 proteins showed enrichment relative to a Siglec-5 control. Six proteins—CD47, CD59, CD73, ITGB6, ITGA3, and ITGB1—were significantly overexpressed in PAAD tissues compared to normal tissues in the TCGA dataset. (c) Response curves showing interactions between Siglec-10 and the six glycoproteins measured by surface plasmon resonance (SPR). Two concentrations (1000 nM, green; 100 nM, red) were tested for all glycoproteins, while ITGA3 was also tested at 300 nM (green) and 30 nM (red). (d) Binding of the SNA lectin (specific for sialic acid) to ITGA3 and ITGB1 recombinant glycoproteins was measured by a lectin array. Sialidase-treated glycoproteins (red bars) showed significantly reduced binding compared to untreated glycoproteins (blue bars). Unpaired t-tests were used for statistical analyses. (e) SPR response curves comparing the binding of intact (sialylated) ITGA3 and desialylated ITGA3 to immobilized Siglec-10. (f) SPR response curves comparing the binding of intact (sialylated) ITGB1 and desialylated ITGB1 to immobilized Siglec-10.

Article Snippet: PDAC cells were incubated with anti-human ITGA3 and ITGB1 antibodies (Clone ASC-1 and TS2/16, respectively, from Thermo Fisher Scientific) at a concentration of 1 μg/mL in 1× PBS at 4°C for 15 minutes.

Techniques: Recombinant, Control, Generated, Mass Spectrometry, Binding Assay, SPR Assay

Journal: bioRxiv

Article Title: Targeting Siglec-10/α3β1 Integrin Interactions Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1101/2025.05.06.652455

Figure Lengend Snippet: (a) Flow cytometric analysis of ITGA3 expression on PDAC cell lines (HS766T, AsPC-1, and PANC-1) along the x-axis. (b) Comparative expression of ITGA3 in PAAD tissues versus normal tissues in the TCGA dataset. Unpaired t tests. * = P<0.05. (c) Flow cytometric analysis of ITGB1 expression on PDAC cell lines (HS766T, AsPC-1, and PANC-1) along the x-axis. (d) Comparative expression of ITGB1 in PAAD tissues versus normal tissues in the TCGA dataset. Unpaired t tests. * = P<0.05. (e–g) Survival analysis of pancreatic tumor patients in the TCGA dataset showing the correlation between (e) CD24, (f) ITGA3, and (g) ITGB1 expression on overall survival. (h) Single-cell RNA-seq analysis of publicly available datasets using BBrowser software. Clusters representing distinct cell types were categorized into normal (blue clusters) and PDAC (orange clusters). (i) ITGA3 expression levels in epithelial cell clusters from normal tissues (blue bar) and PDAC tissues (orange bar). The intensity of red represents the expression of ITGA3, with darker shades indicating higher expression levels. Mann Whitney u test was used for statistical analysis. (j) Pathway analysis comparing ITGA3 hi and ITGA3 low epithelial cells in PDAC tissues to identify upregulated and downregulated pathways. (k) ITGB1 expression levels in epithelial cell clusters from normal tissues (blue bar) and PDAC tissues (orange bar). The intensity of red represents the expression of ITGB1, with darker shades indicating higher expression levels. Mann Whitney u test was used for statistical analysis.

Article Snippet: PDAC cells were incubated with anti-human ITGA3 and ITGB1 antibodies (Clone ASC-1 and TS2/16, respectively, from Thermo Fisher Scientific) at a concentration of 1 μg/mL in 1× PBS at 4°C for 15 minutes.

Techniques: Expressing, RNA Sequencing, Software, MANN-WHITNEY

Journal: bioRxiv

Article Title: Targeting Siglec-10/α3β1 Integrin Interactions Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1101/2025.05.06.652455

Figure Lengend Snippet: (a) Schematic representation of the experimental setup to enrich for ITGA3 low or ITGB1 low PDAC cells. Columns coated with anti-ITGA3 or anti-ITGB1 antibodies were used to isolate ITGA3 low or ITGB1 low cells (cells passing through the columns without binding), respectively. Cells passing through uncoated columns (expressing high levels of ITGA3 or ITGB1) were used as controls. (b) Representative flow cytometry analysis showing the expression of ITGA3 (left) or Siglec-10 ligands (right) expression levels on MIA PaCa-2 cells, depleted from ITGA3 hi cells (ITGA3 low ) or not (control cells; Ctrl cells). (c) Schematic representation of the experimental setup to test the phagocytic capacity of monocyte-derived macrophages targeting control cells (enriched with ITGA3 hi cells) or ITGA3 low cells (n = 4–6). (d-e) Phagocytosis of control and ITGA3 low MIA PaCa-2 (c) or PANC1 (d) PDAC cells by macrophages from different donors. Top: Representative images where increased red indicates higher phagocytosis. Bottom left: Live imaging results (each symbol represents data from one donor). Bottom right: Area under the curve (AUC) data compiled from multiple donors. Statistical significance was assessed using ratio paired t-tests. (f) Schematic representation of the experimental setup to test the phagocytic capacity of monocyte-derived macrophages targeting control cells (enriched with ITGB1 hi cells) or ITGB1 low cells (n = 4–6). (g-h) Phagocytosis of control and ITGB1 low MIA PaCa-2 (f) or PANC1 (g) PDAC cells by macrophages from different donors. Top: Representative images showing phagocytosis (increased red indicates higher phagocytosis). Bottom left: Live imaging results (each symbol represents data from one donor). Bottom right: AUC data compiled from multiple donors. Statistical significance was assessed using ratio paired t-tests.

Article Snippet: PDAC cells were incubated with anti-human ITGA3 and ITGB1 antibodies (Clone ASC-1 and TS2/16, respectively, from Thermo Fisher Scientific) at a concentration of 1 μg/mL in 1× PBS at 4°C for 15 minutes.

Techniques: Binding Assay, Expressing, Flow Cytometry, Control, Derivative Assay, Imaging

Journal: bioRxiv

Article Title: Targeting Siglec-10/α3β1 Integrin Interactions Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1101/2025.05.06.652455

Figure Lengend Snippet: (a) Model illustrating the mechanism of Siglec-10-mediated inhibition of macrophage phagocytosis. Siglec-10 expressed on macrophages binds to its glycan ligands on PDAC cells, which are present on multiple proteins such as ITGA3, ITGB1, and CD24. This interaction induces inhibitory signaling in macrophages, suppressing phagocytosis (left panel). Blocking Siglec-10 with an antibody prevents inhibitory signaling, thereby enhancing macrophage phagocytic ability (right panel). (b) Screening of recombinant antibodies from the top clones for Siglec-10 binding using ELISA. Binding to immobilized Siglec-10 (blue) and Siglec-5 (gray) proteins is shown. (c) Flow cytometric analysis of clone selectivity, showing binding to CHO-K1 cells expressing either Siglec-10 (blue) or Siglec-5 (gray). (d) Area under the curve (AUC) analysis of an in vitro phagocytosis assay to screen the ability of Siglec-10 antibody clones, as well as the commercially available antibodies against CD24 and Siglec-10, to enhance the phagocytic activity of macrophages against AsPC-1 PDAC cells. (e) AUC analysis of the in vitro phagocytosis assay for the top-performing Siglec-10 antibody clone using macrophages differentiated from the monocytes of four donors. Statistical significance was determined using Friedman’s ANOVA test. (f) Time-course analysis of the in vitro phagocytosis assay for the top Siglec-10 blocking antibody clone (68A11A1, blue) compared to the isotype control (gray). Results are based on n=4 independent experiments. (g) ELISA-based binding analysis of the 68A11A1 recombinant antibody to immobilized recombinant Siglec-10 and Siglec-5 proteins at different dilutions. (h) Evaluation of the ability of anti-CD24 or recombinant Siglec-10 antibody (clone 68A11A1) to enhance macrophage-mediated phagocytosis of several PDAC cell lines (AsPC-1, MIA PaCa-2, and PANC-1). Phagocytosis was normalized to the isotype control for each antibody and conducted using macrophages differentiated from monocytes of 5–8 healthy donors. Each symbol represents data from an individual donor, and statistical analyses were performed using ratio paired t-tests compared to the isotype control. On the right, a representative image of the killing assays is shown (red indicates increased killing).

Article Snippet: PDAC cells were incubated with anti-human ITGA3 and ITGB1 antibodies (Clone ASC-1 and TS2/16, respectively, from Thermo Fisher Scientific) at a concentration of 1 μg/mL in 1× PBS at 4°C for 15 minutes.

Techniques: Inhibition, Glycoproteomics, Blocking Assay, Recombinant, Clone Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Phagocytosis Assay, Activity Assay, Control