Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs bind to native ADAM8. Flow cytometry was performed using HEK293 cells expressing full-length ADAM8 (HEK-A8) vs. an empty vector control DNA (HEK-EV) and decreasing antibody (Ab) concentrations. Mean Fluorescent Intensity (MFI) indicates extent of binding of each Ab.

Article Snippet: This library was transiently transfected into HEK293 cells and expression confirmed using flow cytometry with an anti-ADAM8 Ab (Control A8 Ab, MAB10311, R&D Systems, RRID: AB_2273524), whose binding [within the cysteine-rich domain and EGF-like domain (CRD-ELD) region of ADAM8] is not affected by the mutations.

Techniques: Flow Cytometry, Expressing, Plasmid Preparation, Control, Binding Assay

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs bind with high affinity to recombinant human ADAM8 (rHuADAM8). The half-maximal effective concentration (EC50) obtained in ELISA and binding kinetics (k a , k d , and K D ) obtained through Biacore studies for each ADP are shown.

Article Snippet: This library was transiently transfected into HEK293 cells and expression confirmed using flow cytometry with an anti-ADAM8 Ab (Control A8 Ab, MAB10311, R&D Systems, RRID: AB_2273524), whose binding [within the cysteine-rich domain and EGF-like domain (CRD-ELD) region of ADAM8] is not affected by the mutations.

Techniques: Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs bind to five epitope clusters on ADAM8. Diagram indicating the epitope clusters for ADP binding on human ADAM8, and their partial overlap identified based on epitope binning using competitive ELISA.

Article Snippet: This library was transiently transfected into HEK293 cells and expression confirmed using flow cytometry with an anti-ADAM8 Ab (Control A8 Ab, MAB10311, R&D Systems, RRID: AB_2273524), whose binding [within the cysteine-rich domain and EGF-like domain (CRD-ELD) region of ADAM8] is not affected by the mutations.

Techniques: Binding Assay, Competitive ELISA

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs have potent in vitro dual metalloprotease and disintegrin (MP and DI) domain inhibitory activity. ( A ) ADAM8 MP activity was assessed in the presence of each ADP vs. its isotype-matched control IgG by measuring the release of soluble CD23 from the surface of HEK293 cells ectopically overexpressing both ADAM8 and CD23. After overnight antibody (Ab) treatment, conditioned cell media was tested for cleaved CD23 via the detection of its HA-tag in immunoblotting; images were quantified using densitometry. ( B ) ADAM8 DI activity was evaluated in the presence of each ADP vs. control IgG in assays measuring binding of α9β1-Integrin-expressing CHO cells to plates coated with recombinant human ADAM8 (rHuADAM8). Mean MP and DI activity level ± standard deviation (S.D.) from 3 independent experiments is graphed in A and B, respectively. The dashed line represents the level of activity in the presence of MAB1031. Ab-mediated percent inhibition of activity in each case was calculated as a decrease from control IgG levels, which were set to 100%. Mean percent inhibition for each ADP and for MAB1031 ± S.D. is given in red over the respective activity bars.

Article Snippet: This library was transiently transfected into HEK293 cells and expression confirmed using flow cytometry with an anti-ADAM8 Ab (Control A8 Ab, MAB10311, R&D Systems, RRID: AB_2273524), whose binding [within the cysteine-rich domain and EGF-like domain (CRD-ELD) region of ADAM8] is not affected by the mutations.

Techniques: In Vitro, Activity Assay, Control, Western Blot, Binding Assay, Expressing, Recombinant, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs with in vivo anti-tumor activity bind to the ADAM8 DI. ( A ) ADP2, ADP3, or ADP13 binding to HEK293 cells expressing full-length ADAM8 (HEK-A8) vs. remnant form ADAM8 (HEK-REM) was assessed by flow cytometry; HEK293 cells expressing empty vector DNA (HEK-EV) were used as a negative control. Representative histograms of three independent runs are shown. ( B ) Schematic representation of the ADAM8 constructs used in part A, with domain information, amino acid (AA) numbers, and immunogen used for ADP generation indicated. The broad epitope region for ADP2, ADP3, and ADP13 binding to ADAM8, identified by the flow cytometry analysis in part A, is indicated (striped box). ADAM8 domains: PRO—prodomain; MP—metalloproteinase; DI—disintegrin; CRD—cysteine-rich; ELD—EGF-like; TM—transmembrane; CYTO—cytoplasmic. ( C ) Three-dimensional model of the predicted ADAM8 extracellular structure (residues 195-647, including MP, DI, CRD, and ELD) using the crystal structure of ADAM22 as template and Swiss-model software (2003). Regions of ADP2, ADP3, and ADP13 binding, including overlapping sequences, identified through hydrogen/deuterium exchange–mass spectrometry (HDX-MS) analysis are indicated. MP with active catalytic site, DI with integrin-binding region, and hypervariable region (HVR) of CDR are shown.

Article Snippet: This library was transiently transfected into HEK293 cells and expression confirmed using flow cytometry with an anti-ADAM8 Ab (Control A8 Ab, MAB10311, R&D Systems, RRID: AB_2273524), whose binding [within the cysteine-rich domain and EGF-like domain (CRD-ELD) region of ADAM8] is not affected by the mutations.

Techniques: In Vivo, Activity Assay, Binding Assay, Expressing, Flow Cytometry, Plasmid Preparation, Negative Control, Construct, Software, Mass Spectrometry

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: Amino acids (AAs) within the ADAM8 DI mediating ADP2 and ADP13 binding. ( A ) AA residues important for ADP2 and ADP13 binding to ADAM8 were identified using alanine (ALA) scanning mutagenesis plus flow cytometry. Mean binding reactivity (in duplicate samples) of ADP2 or ADP13 antigen−binding fragments (Fabs) to ADAM8 protein mutated at the indicated residues (mutation) within the MP and DI vs. binding of a positive control ADAM8 Ab (Control A8 Ab), which binds outside the MP and DI regions and is therefore unaffected, is presented as a percentage of binding to wild−type (WT) ADAM8. The range of binding reactivity (maximum–minimum) in each case is indicated in parentheses. AAs identified as critical for binding (i.e., those for which Control A8 Ab binding was >70% of WT but test Ab binding was <20% of WT binding) are shown in red boxes. Blue boxes show residues of secondary importance, i.e., AAs in close proximity to critical residues whose mutation led to a substantial (although not <20% of WT) reduction in binding. Epitope AAs for ADP2 ( B ) and ADP13 ( C ) Fab binding, identified through mutagenesis, are indicated on a crystal structure model of the ADAM8 ectodomain based on the structure of vascular apoptosis−inducing protein−1.

Article Snippet: This library was transiently transfected into HEK293 cells and expression confirmed using flow cytometry with an anti-ADAM8 Ab (Control A8 Ab, MAB10311, R&D Systems, RRID: AB_2273524), whose binding [within the cysteine-rich domain and EGF-like domain (CRD-ELD) region of ADAM8] is not affected by the mutations.

Techniques: Binding Assay, Mutagenesis, Flow Cytometry, Positive Control, Control

Journal: Frontiers in Microbiology

Article Title: Inhibition of HIV-1 release by ADAM metalloproteinase inhibitors

doi: 10.3389/fmicb.2024.1385775

Figure Lengend Snippet: HIV infection transiently induced ADAM expressions. (A–C) Heatmap showing differential gene expression analyses of interferon response and antiviral genes (A) , cell cycle related genes (B) , and various ADAM genes (C) from next generation whole cell genome RNA sequencing. B1,B2,U1,U2, BB1,BB2 refer to HIV-1 BAL infected (B1,B2), uninfected (U1,U2), and infected samples in the presence of 50 μM BB-94 (BB1, BB2) from cells of two individual healthy donors. The heatmaps are color coded from red (upregulated genes) to blue (down regulated genes). The statistical analyses were done using two-way ANOVA with p -values are <0.00001 (****). (D) ADAM expression. RNA sequencing data showing the gene expression levels of various ADAMs in CD4 T cells. ADAM8, 9, 10, 15, 17, and 19 expressions are significant by RNA sequencing. The gene expressions are shown as FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced). The overall ADAM expression profiles exhibited similar patterns in PBMC and CD4 T cells. Data include both infected and uninfected samples. (E) Western blot analyses of ADAM8, 10, 15, 17 and 19 expressions in uninfected PBMC. (F) Wester blot detection of ADAM10 and 17 on day 0 (D0) and 3 (D3) HIV-1 infected versus uninfected samples with and without 50 μM BB-94 treatment. (G) RT-PCR analyses on the relative expression of ADAM9, 10 and 17 on day 3 and 6 from uninfected and HIV-1 BAL infected PBMC in the presence and absence of BB-94. The expression of all three ADAMs are upregulated in HIV infected cells. (H) Relative expressions of ADAM9, 10 and 17 in day 3 and 6 post infection as analyzed by RT-PCR. ADAM expressions were elevated in HIV infected samples on day 3 but returned to the uninfected levels on day 6 of post infection. Statistics are calculated using multiple t -tests with p -values indicated above their data. p -values are *<0.05, **<0.01, ***<0.001.

Article Snippet: SDS samples of protein extracts were electrophoresed, transferred to PVDF (IB34002, Thermo, USA) membranes, blocked with 5% non-fat dry milk (catalog 1,706,404, BioRad, USA) containing 0.1% Tween20 (catalog P1379, Sigma, USA) for 2 h at RT, and incubated overnight at 4°C with antibodies against ADAM8 (1:1000 dilution, catalog MAB10311, R&D systems), ADAM10 (1:1000 dilution, catalog ab1997, Abcam, USA), ADAM15 (1:1000 dilution, catalog MAB935, R&D systems), ADAM17 (1:1000 dilution, catalog PA5-11572, Invitrogen, USA), ADAM19 (1:1000 dilution, catalog ab191457, Abcam), or beta-Actin (1:1000 dilution, catalog MA5-15739, Invitrogen, USA), respectively.

Techniques: Infection, Gene Expression, RNA Sequencing, Expressing, Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: miR-720 is a downstream target of an ADAM8-induced ERK signaling cascade that promotes the migratory and invasive phenotype of triple-negative breast cancer cells

doi: 10.1186/s13058-016-0699-z

Figure Lengend Snippet: ADAM8 DI domain induces expression of miR-720 via β1-integrin signaling. a , b HEK-293 cells were transfected in six-well plates with 2 μg of either pcDNA3.1 myc-his vector ( EV-3.1 ), or vectors expressing ADAM8 ( WT-3.1 or EQ-3.1 ) for 48 h. WCEs and RNA were then collected. Samples of WCEs were subjected to Western blotting for ADAM8 and β-actin, as in Fig. . A representative blot is shown ( n = 3) ( a ). RNAs were subjected to RT-qPCR for measurement of miR-720 levels and values presented relative to the control condition (EV-3.1), which is set to 1 (mean ± SD from three independent experiments), as above ( b ). c , d HEK-293 cells were transfected as above with EV DNA or with vectors expressing ADAM8 WT or remnant form ( Rem. ) for 24 h and 48 h. WCEs harvested 48 h after transfection were analyzed for ADAM8 and β-tubulin. A representative blot is shown ( n = 3) ( c ). RNA was subjected to RT-qPCR for measurement of miR-720 levels. EV control condition is set to 1 (mean ± SD from three independent experiments) ( d ). e HEK-293 cells were transfected in 12-well plates with a vector expressing the ADAM8 remnant form ( Rem. ). After 24 h, the transfected cells were treated with 20 μg/ml ADAM8 antibody MAB10311, which targets the CRD/ELD domains and inhibits DI activity , or isotype-matched control IgG1 for another 24 h. RNA was extracted and subjected to RT-qPCR analysis for miR-720 levels. Control condition (EV + IgG1) is set to 1 (mean ± SD from three independent experiments). f MDA-MB-231 cells were treated with 10 or 20 μg/ml β1-integrin ( Anti-β1-integ. ) antagonist antibody or isotype-matched control IgG2A for 24 h, and miR-720 levels determined by RT-qPCR analysis. Control condition (IgG2A) is set to 1 (mean ± SD from three independent experiments). ** P < 0.01, *** P < 0.001, Student’s t test. Not sig. not significant, Rel. relative

Article Snippet: The anti-ADAM8 antibody MAB10311 and its control isotype-matched IgG1 (MAB002) were from R&D Systems.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Activity Assay

Journal: Breast Cancer Research : BCR

Article Title: miR-720 is a downstream target of an ADAM8-induced ERK signaling cascade that promotes the migratory and invasive phenotype of triple-negative breast cancer cells

doi: 10.1186/s13058-016-0699-z

Figure Lengend Snippet: ADAM8 DI domain induces expression of miR-720 via β1-integrin signaling. a , b HEK-293 cells were transfected in six-well plates with 2 μg of either pcDNA3.1 myc-his vector ( EV-3.1 ), or vectors expressing ADAM8 ( WT-3.1 or EQ-3.1 ) for 48 h. WCEs and RNA were then collected. Samples of WCEs were subjected to Western blotting for ADAM8 and β-actin, as in Fig. . A representative blot is shown ( n = 3) ( a ). RNAs were subjected to RT-qPCR for measurement of miR-720 levels and values presented relative to the control condition (EV-3.1), which is set to 1 (mean ± SD from three independent experiments), as above ( b ). c , d HEK-293 cells were transfected as above with EV DNA or with vectors expressing ADAM8 WT or remnant form ( Rem. ) for 24 h and 48 h. WCEs harvested 48 h after transfection were analyzed for ADAM8 and β-tubulin. A representative blot is shown ( n = 3) ( c ). RNA was subjected to RT-qPCR for measurement of miR-720 levels. EV control condition is set to 1 (mean ± SD from three independent experiments) ( d ). e HEK-293 cells were transfected in 12-well plates with a vector expressing the ADAM8 remnant form ( Rem. ). After 24 h, the transfected cells were treated with 20 μg/ml ADAM8 antibody MAB10311, which targets the CRD/ELD domains and inhibits DI activity , or isotype-matched control IgG1 for another 24 h. RNA was extracted and subjected to RT-qPCR analysis for miR-720 levels. Control condition (EV + IgG1) is set to 1 (mean ± SD from three independent experiments). f MDA-MB-231 cells were treated with 10 or 20 μg/ml β1-integrin ( Anti-β1-integ. ) antagonist antibody or isotype-matched control IgG2A for 24 h, and miR-720 levels determined by RT-qPCR analysis. Control condition (IgG2A) is set to 1 (mean ± SD from three independent experiments). ** P < 0.01, *** P < 0.001, Student’s t test. Not sig. not significant, Rel. relative

Article Snippet: The anti-ADAM8 antibody MAB10311 and its control isotype-matched IgG1 (MAB002) were from R&D Systems.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Control, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A ADAM8 mRNA expression in samples from breast tumor and normal breast tissue was analyzed using the Oncomine microarray database. Pooling of 14 analyses from six different microarray studies shows ADAM8 is one of the more highly expressed genes in breast cancer versus normal tissue. P = 0.025, Student's t -test. B, C ADAM8 protein levels were measured by ELISA in samples from adjacent normal breast tissue (NBT), fibroadenoma (FA) and primary breast carcinoma (PBC) (B), and in serum of patients with either benign or malignant breast disease (C). * P < 0.0001, Kruskal–Wallis test (B); * P = 0.034, Mann–Whitney U -test (C). D ADAM8 mRNA expression was analyzed across the different molecular breast cancer subtypes in the van de Vijver microarray dataset, which includes 295 primary breast tumors from normal-like (Normal), luminal A (Lum A), luminal B (Lum B), HER2, and basal-like (Basal) subtypes (van de Vijver et al , ). * P < 0.0001 for Basal versus other groups, Kruskal–Wallis test. E Representative pictures of ADAM8 staining analyzed by immunohistochemistry in adjacent normal epithelial tissue and primary TNBC samples from 50 patients or areas of microinvasion. Percentages of ADAM8-positive samples are given. F, G Kaplan–Meier curves show the percentage of disease-free survival (F) and overall survival (G) for 295 patients with primary breast cancer stratified based on ADAM8 mRNA levels using the 75th percentile. P < 0.0001, Log-rank test (RR, relative risk, CI, confidence interval).

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Expressing, Microarray, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A Schematic representation of ADAM8 protein with its domains, processed forms and molecular weights indicated. CYS-Rich: cysteine-rich, EGF: EGF-like, TM: transmembrane domains. B Whole-cell extracts (WCEs) from human non-tumoral MCF-10A cells and the indicated TNBC cell lines were examined by WB for ADAM8 expression (Millipore antibody), and for β-Actin as a loading control. A representative blot is shown ( n = 3). All lanes were from the same gel, but cut to re-align as indicated by the vertical line. ADAM8 forms and MW markers are indicated. ns: non-specific band. MDA-231: MDA-MB-231. MDA-468: MDA-MB-468. C Cells were grown in adherent (2D) or suspension cultures on ultra low-attachment plates (3D) for 48 h. In suspension, MDA-MB-231 and Hs578T cells form spheres. Bar: 100 μm. D, E MDA-MB-231 (D) and Hs578T (E) cells were transfected with either Control siRNA (siCtrl) or two specific ADAM8 siRNAs (siA8) for 24 h. Cells were then plated in 2D-or 3D-culture as described in (C). WCEs were subjected to WB for ADAM8 using only LSBio antibody (D), or using both LSBio and Millipore antibodies (E). As a loading control, Tubulin (D) or β-Actin (E) was used. Representative blots are shown ( n = 3).

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Expressing, Control, Suspension, Transfection

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A–D Cells were transfected with siRNAs as in Fig D and E for 24 h, and tested for colony formation in soft agar (A), cell migration (B), invasion (C), and 3D-Matrigel outgrowth (D). For soft agar assays, cells were grown for 8-12 days and colonies >20 μm diameter in 3 wells/condition were counted with ImageJ software. A representative of two experiments with similar results is shown. * P = 0.002, ** P = 1.9E-6, # P = 1.3E-5, ## P = 1.0E-7, Student's t- test (A). Migration (B) and invasion (C) assays were performed for 24 h using Transwells without or with precoating of Matrigel, respectively. Control condition (siCtrl) is set to 100% (mean ± s.d. from three independent experiments). * P = 0.004, ** P = 5.3E-4, # P = 1.8E-4, ## P = 5.5E-5, Student's t- test (B). * P = 0.001, ** P = 9.3E-4, # P = 8.7E-6, ## P = 4.6E-5, Student's t- test (C). For Matrigel outgrowth assays, colonies formed after 5-7 days were photographed at 20× magnification. Experiments were done twice with similar results. Bars: 100 μm (D). E WCEs from MDA-MB-231 cells and in vivo derived LM1 and LM2 lines, either untransfected (left panel) or transfected with siCtrl or siA8 for 48 h (right panels), were subjected to WB for ADAM8 (LSBio antibody). A representative blot is shown ( n = 3). F Twenty-four h after siRNA transfection, LM1 and LM2 cells were subjected to a 3D-Matrigel outgrowth assay, as in (D), except that colonies were photographed after 4 days. Experiments were done twice with similar results. Bars: 100 μm.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Transfection, Migration, Software, Control, In Vivo, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A–D Stable ADAM8 KD (shA8) clones were characterized in vitro . ADAM8 expression in WCEs from two shA8 clones (shA8-17 and shA8-20) were compared with two shCtrl clones (shCtrl-3 and shCtrl-5) using WB (LSBio antibody) (A). Clones were grown in 2D-cultures for 48 h and subjected to an ATP assay. NS: non significant, Student's t -test, n = 3 (B). Migration assays were performed as described in Fig B. * P = 6.8E-14, Student's t- test, n = 3 (C). Matrigel outgrowth assays were performed as in Fig D. Experiments were done twice with similar results. Bar: 100 μm (D). E–H MDA-MB-231 derived shCtrl-3 and shA8-20 cells were injected into the MFP of female mice ( n = 7/group). Tumor volume was measured twice a week (mean ± s.e.m.). * P = 1.4E-6, ** P = 9.8E-8, # P = 3.0E-5, ## P = 9.7E-7, † P = 1.7E-5, ‡ P = 5.1E-6, § P = 2.0E-7, Student's t -test (E). At the end of the experiment, tumors were photographed and weighed (mean ± s.e.m.). * P = 5.6E-8, Student's t -test. Bar: 1 cm (F). Blood was collected by cardiac puncture and GFP-positive CTCs were detected by flow cytometry. Average CTC count ± s.d. is given per μl of blood. * P = 0.006, Student's t -test (G). Presence of brain metastases was examined by fluorescent microscopy ( n = 6/group). Representative photographs are shown. Bars: 1 mm (H). I ADAM8 expression in distant metastases of breast cancer patients ( n = 56) was analyzed by immunohistochemistry. Representative pictures and percentages of ADAM8-positive samples are presented.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Clone Assay, In Vitro, Expressing, ATP Assay, Migration, Derivative Assay, Injection, Flow Cytometry, Microscopy, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A Sixteen h after plating, cells were cultured under normoxic (−) or hypoxic (+, 1% O2) conditions for 24 h (MDA-MB-231 and Hs578T) or 6 h (shCtrl-3 and shA8-20 clones). WCEs were subjected to WB for ADAM8 (LSBio antibody). Representative blots are shown ( n = 3). B ADAM8 expression in mouse mammary tumors derived from shCtrl-3 or shA8-20 cells was analyzed by immunohistochemistry. H&E staining was performed in parallel. Representative panels are shown ( n = 7/group). Bar: 100 μm. C Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections from shCtrl-3 and shA8-20 groups ( n = 7/group). Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (3 peritumoral hot spots/slide). P: Peritumoral area, T: Tumor. Bar: 100 μm. * P = 0.01, Student's t -test. D Pearson's pairwise correlation plot shows a significant positive correlation between ADAM8 and CD31 (PECAM1) mRNA expression in tumors from patients with basal-like breast cancer (GenExMiner microarray database). r: correlation ratio. P < 0.0001, Student's t -test. E, F HUVECs were subjected to tube formation assays in the presence of conditioned medium from the indicated shA8 and shCtrl clones, or obtained in absence of tumor cells (−). Values for branch points and closed networks (polygons) are given as averages of nine fields ± s.d. Branch point: * P = 1.3E-6 versus shCtrl-3, * P = 3.2E-9 versus shCtrl-5, ** P = 6.9E-29; Polygons: * P = 5.2E-6 versus shCtrl-3, * P = 1.3E-6 versus shCtrl-5, ** P = 6.2E-24; Student's t -test; n = 4 (E). Representative images from 4 independent experiments are shown. Bar: 30 μm (F). G Conditioned media from two shCtrl and two shA8 clones were subjected to a Human Angiogenesis antibody array. Expression levels of the detected proteins were quantified using ImageJ and the angiogenesis mediators significantly downregulated by more than 2-fold in shA8 clones are presented as mean of the two clones ± s.d. Fold change (F.C.) and P -values are given, Student's t -test. H Conditioned serum-free medium from shCtrl and shA8 clones was analyzed by WB for VEGF-A. A representative blot is shown ( n = 3). I VEGF-A in the conditioned serum-free medium from shCtrl-3 or shA8-20 clones transfected with the indicated ADAM8 forms or empty vector (EV) DNA was assessed by WB (lower panel). The quantification of average levels from 3 experiments is presented as percent relative to the shCtrl set to 100%.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Cell Culture, Clone Assay, Expressing, Derivative Assay, Immunohistochemistry, Staining, Immunohistochemical staining, Microarray, Ab Array, Transfection, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A Blood was drawn using the submandibular collection method from 4 mice/group on the indicated days after tumor cell implantation into the MFP and subjected to flow cytometry to measure GFP-positive CTCs. Average count for the 4 mice ± s.d. per μl of blood is given for each time point. * P = 0.03, # P = 0.05, † P = 0.02, Student's t -test. B Cells were incubated, in duplicate, on a HUVEC monolayer or in empty wells. Attached cells were counted in three fields per well. Mean ± s.d. from three independent experiments. NS, non significant, * P = 1.6E-6, Student's t -test. C Cells were subjected to an overnight transendothelial migration assay on HUVECs. Mean ± s.d. from three independent experiments. * P = 1.7E-5, Student's t -test. D, E Cells were plated on fibronectin-coated coverslips, stained with antibodies against active β1-integrin, vinculin or phosphotyrosine (focal adhesions markers) and phalloidin-488 (F-actin). Confocal microscopy images show adhesion protein channel (red) or merge with F-actin (green). Bar: 10 μm (D). Signal intensity for active β1-integrin and vinculin was determined using ImageJ software (arbitrary units). Mean intensity ± s.d. from >20 cells over 2 experiments. * P = 1.4E-4, NS, non significant, Student's t -test (E). F Adhesion of shCtrl-3 cells on HUVECs was assessed as in (B) with prior incubation of shCtrl-3 cells with an antagonist β1-integrin antibody (+) or a control isotype (−). Mean ± s.d. from three independent experiments. * P = 3.0E-5, Student's t -test. G, H Adhesion (G) and transendothelial migration (H) of shCtrl-3 cells on HUVECs was assessed as in B and C, respectively. Prior to the assays, shCtrl-3 cells were incubated either with monoclonal antibodies targeting the ectodomain of ADAM8 (Mab10311 or Mab1031) or an appropriate isotype control (IgG1 or IgG2B, respectively). The transmigration assay was performed for 9 h. Mean ± s.d. from three independent experiments. * P = 0.001, # P = 5.0E-4, Student's t -test (G). * P = 6.3E-7, # P = 4.0E-7, Student's t -test (H). I Active β1-integrin expression was assessed by immunohistochemistry in mouse mammary tumors (shCtrl-3 and shA8-20) and contralateral mammary glands (shCtrl-3) ( n = 7/group). P: Peritumoral area, T: Tumor. Bar: 100 μm.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Flow Cytometry, Incubation, Migration, Staining, Confocal Microscopy, Software, Control, Bioprocessing, Transmigration Assay, Expressing, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A–E MDA-MB-231 shCtrl-3 cells were injected into the MFP of female mice. Animals were treated with either 0.5 mg/kg anti-ADAM8 (anti-A8, Mab1031, n = 9) or isotype-matched control (IgG2B, n = 8) in i.p. injection twice weekly. Tumor volume was measured on the indicated days (mean ± s.e.m.). * P = 0.02 (day 11), # P = 0.01 (day 13), † P = 2.1E-4 (day 17), ‡ P = 1.5E-4 (day 20), Student's t -test (A). At the end of the experiment, tumors were weighed (mean ± s.e.m.). * P = 5.8E-4, Student's t -test (B) and the presence of brain metastases was examined by fluorescent microscopy (C). Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections. Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (5 peritumoral hot spots/slide) and representative pictures are shown (D). P: Peritumoral area, T: Tumor. Bar: 100 μm. VEGF-A levels in the tumor extracts were determined by WB and normalized to Coomassie staining (E). * P = 0.04 (D), * P = 0.03 (E), Student's t -test. F–G Scheme of experimental design (F). Metastases to the brain and lungs were examined by fluorescent microscopy. Representative images and frequency of metastases (percentage of animals positive per group: IgG2B, n = 8; anti-A8, n = 9) are presented (G). Bar: 250 μm.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Injection, Control, Microscopy, Immunohistochemical staining, Staining

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: When solid tumors reach a few millimeters in diameter, hypoxic stress is induced, which leads to ADAM8 induction resulting in strong pro-angiogenic signaling, in part via VEGF-A release into the extracellular compartment, and endothelial cell recruitment. ADAM8 also promotes β1-integrin activation on tumor cells needed for their adhesion onto and transmigration through the blood vessel wall, which supports dissemination of CTCs and development of metastases. Importantly, here we demonstrate that if the induction of ADAM8 is blocked or its activity inhibited with antibody, there is an insufficient angiogenic response, leading to tumor mass dormancy or slowing of growth, as well as to a striking reduction of CTCs and metastases.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Activation Assay, Transmigration Assay, Activity Assay