Journal: Journal of Extracellular Vesicles

Article Title: αvβ1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin‐3

doi: 10.1002/jev2.12234

Figure Lengend Snippet: Isolation and characterization of EVs from breast cancer cells. (a) Schema of isolation strategy to obtain breast cancer cell‐derived EVs from conditioned medium using a two‐step EV isolation regimen combining flotation ultracentrifugation and fast protein size‐exclusion liquid chromatography (FPLC‐SEC). Purified EVs were combined from SEC fraction 8 to 14 and concentrated using a filter unit with 100‐kDa cut‐off. (b) Separation of EVs from vesicle‐free proteins using FPLC‐SEC, indicated by the concentrations of EVs as determined using nanoparticle tracking analysis, and the concentration of free proteins, determined using BCA assay and absorbance at 280 nm in each fraction of the FPLC‐SEC. (c) Western blot analysis of proteins in CA1a cells and purified EVs. (d) Flow cytometry analysis of CD63 in purified EVs. (e) Representative images of EVs before and after FPLC‐SEC (fraction 8–14 were combined), obtained by transmission electron microscopy. Scale bars: 0.5 μm. (f) Size distribution of the isolated EVs, determined by nanoparticle tracking analysis (100x diluted)

Article Snippet: We generated in‐house conjugated anti‐mouse CD63 magnetic beads by incubating streptavidin‐conjugated magnetic beads (Thermo Fisher, USA) with biotin‐labelled anti‐mouse CD63 antibody (Miltenyi Biotec, Germany) with orbital rotation and vibration mixing on a HulaMixer (Thermo Fisher, USA) for 45 min at RT.

Techniques: Isolation, Derivative Assay, Liquid Chromatography, Purification, Concentration Assay, BIA-KA, Western Blot, Flow Cytometry, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: αvβ1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin‐3

doi: 10.1002/jev2.12234

Figure Lengend Snippet: Integrin αv and β1 are more abundant on the surface of EVs from breast cancer cells of highly metastatic origin than EVs from cells of moderately metastatic potential. (a‐b) Flow cytometry analysis of integrin β1 and αv (ITGB1 and ITGAV, respectively) on the surface of EVs from human highly‐metastatic CA1a and poorly‐metastatic MB‐231 breast cancer cells, captured on anti‐human CD63 magnetic beads. Graphs present the average percentage of EV‐bead complexes with positive staining for ITGB1 and ITGAV in CA1a and MB‐231 EVs ± SEM. (c) Western blot analysis of ITGAV and ITGB1 in CA1a and MB‐231 EVs. TSG101 was used as a loading control. (d‐e) Flow cytometry analysis of ITGB1 and ITGAV on the surface of EVs from mouse highly‐metastatic 4T1 and poorly‐metastatic 4TO7 breast cancer cells, captured on anti‐mouse CD63 magnetic beads. Graphs present the average percentage of EV‐bead complexes with positive staining for ITGB1 and ITGAV in 4T1 and 4TO7 EVs ± SEM. Student t‐test ** P ≤ 0.01, *** P ≤ 0.0001

Article Snippet: We generated in‐house conjugated anti‐mouse CD63 magnetic beads by incubating streptavidin‐conjugated magnetic beads (Thermo Fisher, USA) with biotin‐labelled anti‐mouse CD63 antibody (Miltenyi Biotec, Germany) with orbital rotation and vibration mixing on a HulaMixer (Thermo Fisher, USA) for 45 min at RT.

Techniques: Flow Cytometry, Magnetic Beads, Staining, Western Blot, Control

Journal: Journal of Extracellular Vesicles

Article Title: αvβ1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin‐3

doi: 10.1002/jev2.12234

Figure Lengend Snippet: Integrin αv is associated with cancer progression in patients with breast cancer. (a) Scoring scheme and representative images from ITGAV IHC staining of tissue sections from patients with breast cancer. (b) Association of ITGAV expression score with the presence of lymph node metastasis (LM). (c) Association of ITGAV expression score with cancer N stages. (d) Association of ITGAV expression score with locally advanced and metastatic group (stage IIB ‐ IV) and early staged group (I‐IIA). (e) Receiver operating characteristic curve analysis of the diagnosis for locally advanced and metastatic group based on ITGAV expression score. (f) ELISA analysis of ITGAV protein in circulating EVs (number of ITGAV molecules per 10 5 EVs) purified from blood samples of patients with early stage (I and II) and late stage (III and IV) breast cancer. (g) Receiver operating characteristic curve analysis of the diagnosis for early and late‐stage group based on ITGAV level in circulating EVs. Mann‐Whitney test * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. (h) Representative IHC images of tumour sections stained for ITGAV (red) and CD63 (brown) from breast cancer patients. (I) ITGAV‐CD63 colocalization analysis using Chi‐square test

Article Snippet: We generated in‐house conjugated anti‐mouse CD63 magnetic beads by incubating streptavidin‐conjugated magnetic beads (Thermo Fisher, USA) with biotin‐labelled anti‐mouse CD63 antibody (Miltenyi Biotec, Germany) with orbital rotation and vibration mixing on a HulaMixer (Thermo Fisher, USA) for 45 min at RT.

Techniques: Immunohistochemistry, Expressing, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Purification, MANN-WHITNEY, Staining

Journal: Journal of Extracellular Vesicles

Article Title: αvβ1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin‐3

doi: 10.1002/jev2.12234

Figure Lengend Snippet: Integrin αv is enriched in circulating EVs from xenografted mice bearing metastatic breast cancer tumours. (a) Schema of implanting CA1a or MB‐231 cells in the flank of NSG mice for circulating EV analysis and confocal imaging. (b) Flow cytometry analysis of anti‐human CD63 beads incubated with EVs from untreated NSG mouse serum (control, cont.). (c) Flow cytometry analysis of ITGAV on EVs from the serum of NSG mice bearing CA1a tumour, MB‐231 tumour or no tumour in the flank (cont), captured by anti‐human CD63 beads. Serum from two mice in the same group were pooled ( n = 3 paired groups of mice). (d) Representative images and colocalization analysis of ITGAV and CD63 in CA1a and MB‐231 tumours. Xenografted tumour sections were stained for CD63 and ITGAV. Images and 3D projection were obtained using confocal microscopy to identify colocalized staining and calculate the colocalization coefficient index ( n = 6 mice). Student's t‐test, **** P ≤ 0.0001.

Article Snippet: We generated in‐house conjugated anti‐mouse CD63 magnetic beads by incubating streptavidin‐conjugated magnetic beads (Thermo Fisher, USA) with biotin‐labelled anti‐mouse CD63 antibody (Miltenyi Biotec, Germany) with orbital rotation and vibration mixing on a HulaMixer (Thermo Fisher, USA) for 45 min at RT.

Techniques: Imaging, Flow Cytometry, Incubation, Control, Staining, Confocal Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: αvβ1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin‐3

doi: 10.1002/jev2.12234

Figure Lengend Snippet: Galectin‐3 mediates the export of integrin αv and β1 into CA1a EVs. (a) Flow cytometry analysis of ITGB1 on ITGAV + EVs from CA1a cells, captured by anti‐human ITGAV beads. (b) Proteins associated with ITGAV in CA1a cells and MB‐231 cells, identified using immunoprecipitation and mass spectrometry with a biotinylated ITGAV antibody and immobilized streptavidin magnetic beads. Shown in the map are known protein‐protein interactions among the most abundant ITGB1‐ and ITGAV‐associated proteins Yellow nodes indicate proteins with higher enrichment in the ITGAV immunoprecipitation of CA1a cells compared to MB‐231 cells. (c‐d) qRT‐PCR and Western blot analysis of galectin‐1 and ‐3 (Gal‐1/3 encoded by LGALS1/3 ) in CA1a cells transduced with shRNAs targeting Gal‐1/3 or a scrambled (Scr) control shRNA. (e‐f) Flow cytometry analysis of ITGB1 and ITGAV on CA1a EVs after knockdown of Gal‐1 and Gal‐3, captured by anti‐human CD63 beads. (g‐h) Representative confocal microscopy images and colocalization coefficient (CoE) analysis of ITGB1 and ITGAV with CD63 in CA1a cells after knockdown of Gal‐1 and Gal‐3 ( n = 6). (i) H & E staining of lung sections from NSG‐SGM3 mice injected (i.v.) with CA1a cells transduced with the scrambled or Gal‐3 shRNAs, compared to untreated mice. Metastatic nodules are indicated by the arrows. Scale bar: 500 μm. The graph presents the percentage of lung area with metastasis ( n = 4–6). Student's t‐test ** P ≤ 0.01, *** P ≤ 0.001, ns: non‐significant

Article Snippet: We generated in‐house conjugated anti‐mouse CD63 magnetic beads by incubating streptavidin‐conjugated magnetic beads (Thermo Fisher, USA) with biotin‐labelled anti‐mouse CD63 antibody (Miltenyi Biotec, Germany) with orbital rotation and vibration mixing on a HulaMixer (Thermo Fisher, USA) for 45 min at RT.

Techniques: Flow Cytometry, Immunoprecipitation, Mass Spectrometry, Magnetic Beads, Protein-Protein interactions, Quantitative RT-PCR, Western Blot, Transduction, Control, shRNA, Knockdown, Confocal Microscopy, Staining, Injection

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Allergological work-up outcome.

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Example of a positive BAT-FC in patient 6. Gating strategy for basophils: ( A ) doublet exclusion, FSC-H vs. FSC-A; basophils were identified as low SSC-A and CD123 + ( B ) and CD123 + HLA-DR neg cells ( C ). ( D ) The univariate histogram shows a positive BAT determined by an increased percentage of CD63 expression on the patient’s basophils with the highest concentration of oxaliplatin (250 µg/mL). ( E ) The univariate histogram (green) shows a positive BAT determined by increased mean fluorescence intensity (MFI) on the patient’s basophils at 250 µg/mL oxaliplatin; the gray histogram corresponds to the unstimulated cells. ( F ) Table showing the percentage of CD63+, MFI, and SI of CD203c in patient 6. Data were analyzed using FACSDiva software (verion 8.0.2).

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Expressing, Concentration Assay, Fluorescence, Software

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Examples of positive sIgE-microarray, BAT-microarray, and immunofluorescence detection of CD63 upon drug stimulation in patient 6, allergic to oxaliplatin, and patient 21, allergic to carboplatin. Cells were visualized under at ×100 magnification using a Olympus IX70 fluorescent microscope.

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Microarray, Immunofluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: BAT-microarray immunoassay. The y -axis shows the average CD63 expression represented as average z -scores. The x -axis shows the drug concentration: oxaliplatin ( A ) and carboplatin ( B ) 1:100 molar ratio. The standardized fluorescence intensity represented as the average z -score was considered positive if it exceeded three (dotted line).

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Microarray, Expressing, Concentration Assay, Fluorescence