Journal: Journal of Extracellular Vesicles

Article Title: Signal Amplification for Fluorescent Staining of Single Particles in Liquid Biopsies: Circulating Tumour Cells and Extracellular Vesicles

doi: 10.1002/jev2.70167

Figure Lengend Snippet: (A) Characterization of the size distribution of the EVs isolated from GBM2 CTCs by Izon SEC using NTA. Measured total concentration: 0.9 × 10 9 particles/mL. (B) Characterization of the GBM2 EV protein content (CD9 and EGFR) using ELISA. (C) Characterization of the EV tetraspanin expression and distribution using ONI dSTORM imaging system. CD9‐AF488 (yellow dots), CD63‐AF555 (blue dots) and CD81‐AF594 (pink dots) targeted and analysed. Right hand‐side image: zoom‐in on two EVs co‐expressing the three analysed tetraspanins. The numbers reported beside each tetraspanin represent the number of dots detected on each vesicle and are proportional to the amount/expression of that specific tetraspanin on the EV surface. (D) Validation of the EV capture onto a glass substrate. EVs labelled with tdTomato fluorophore were adsorbed overnight on a TB380 glass slide and imaged the day after, after several washing steps. Two different concentrations (Low EVs = [1×] = 3 × 10 7 particles/mL and High EVs = [8×] = 24 × 10 7 particles/mL) tested, and PSB used as a control. EV counts obtained from four FOVs and plotted on the right as mean ± SD. Images obtained with the 100× oil immersion lens (NA 1.45) of a widefield microscope (Nikon 90i) equipped with a cooled CCD camera (Andor Clara DR‐2519) and a 1.6× optical coupler (Nikon Digital Imaging Head).

Article Snippet: The membranes were incubated overnight with primary antibodies (1:500 dilutions in 5% BSA with 0.02% sodium azide prepared in 1× TBST): HSP70 (cat. # 4872, Cell Signalling), CD63 (cat. # NB100‐77913, Novus Biologicals) and CD9 (cat. # 312102, BioLegend).

Techniques: Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Imaging, Biomarker Discovery, Control, Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Signal Amplification for Fluorescent Staining of Single Particles in Liquid Biopsies: Circulating Tumour Cells and Extracellular Vesicles

doi: 10.1002/jev2.70167

Figure Lengend Snippet: Comparison between DS, PSS and the developed TSA staining protocol using GBM2 EVs. (A, D, G) Representative images of the EVs stained using the three different techniques along with the respective control substrates (no EVs, PBS only + CD63 antibodies). (B, E, H) Fluorescent intensities (plotted as integrated intensities over all the pixels of each single EV) and counts of single EVs stained with the three different techniques, calculated considering two FOVs per technique. (C, F, I) Profiles of the EV pixel intensities over a 5‐min period of continuous laser excitation for the three analysed techniques. The four curves correspond to the pixel intensity distributions of the snapshot images captured at time 0, and after 1, 3 and 5 min of laser excitations, respectively.

Article Snippet: The membranes were incubated overnight with primary antibodies (1:500 dilutions in 5% BSA with 0.02% sodium azide prepared in 1× TBST): HSP70 (cat. # 4872, Cell Signalling), CD63 (cat. # NB100‐77913, Novus Biologicals) and CD9 (cat. # 312102, BioLegend).

Techniques: Comparison, Staining, Control

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 and FHRE circulate on extracellular vesicles (EVs) in normal human serum (NHS) and normal mouse serum (MS). (A) Detection of FHR-1 in the EV fraction from NHS (EV NHS ) but not from homozygous FHR-1-deficient (EV ΔFHR-1 ) human serum by western blot analysis. FHR-1 was also absent from the supernatant fractions of both sera. Results shown are representative of three experiments. An uncropped gel is shown in . (B) Tracking of EV NHS and EV ΔFHR-1 by live-cell imaging using CLSM. EVs were stained with anti-FHR-1 antibodies conjugated with Alexa Fluor 647 (red). Bars: 10 µm. (C–F) Size distribution and number of vesicles isolated from EV FHR-1 and EV FHRE determined by DLSM using NanoSight NTA 3.2 software. Graphs in ( C,E ) represent overlays of results from 3 to 4 donors. ( (C) , SEM ± standard error, *p ≤ 0.0255 by unpaired two-tailed t-test, n = 4), and ( (E) , SEM ± standard error, *p ≤ 0.0232 by unpaired two-tailed t-test, n = 3). (G) Tracking by live-cell imaging using CLSM of EV FHR-1 and EV ΔFHR-1 stained with anti-CD9 antibody (vesicle marker) and SYTOX orange (nucleic acid marker), Bars: 10 µm. (H) Size distribution of EVs transporting CD9 and FHR-1 (EV CD9&FHR-1 ). EV FHR-1&CD9 were captured with anti-CD9-coated beads from EV FHR-1 , isolated from 1 mL NHS and analyzed by DLSM using NanoSight NTA 3.2 software. (I) Particle numbers (J) and sizes (K) in fractions obtained by size-exclusion chromatography of EV NHS measured by DLSM. (L) High FHR-1 content in fractions 13–16 determined by ELISA. FHR-1 (red) (M) and FHR-1, and vesicle marker CD9, were in close proximity (red) (N) in atherosclerotic tissues, mainly in blood vessels (stippled lines). Complexes were analyzed by proximity ligation assays using anti-FHR-1 and anti-CD9, and were detected by CLSM. EV FHR-1 carry nucleic acids (orange). Bars = 10 µm.

Article Snippet: FHR-1-transporting EVs were captured using beads coated with monoclonal FHR-1 antibody and were stained with Alexa Fluor 647-labeled anti-human CD9 antibody (1:100; Novus Biological, cat no. NB500-327).

Techniques: Western Blot, Live Cell Imaging, Staining, Isolation, Software, Two Tailed Test, Marker, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Ligation

Journal: Journal of dairy science

Article Title: Yak milk-derived exosomes alleviate lipopolysaccharide-induced intestinal inflammation by inhibiting PI3K/AKT/C3 pathway activation.

doi: 10.3168/jds.2021-20175

Figure Lengend Snippet: Figure 3. Protein–protein interaction network of the CD46 proteins and immune-related proteins and tight junction proteins. Fifteen pro- teins were linked into the network. CDH1 = E-cadherin; OCLN = occluding; TJP1 = ZO-1; LTA = TNF-β. Known interactions are represented:

Article Snippet: The membranes were blocked with 5% skim milk-TBS-Tween20 for 4 h at room temperature and incubated overnight at 4°C with the primary antibodies (diluted 1:1,000 in PBS) against proteins of membrane CD46 (cat. # NB500–301, Novus Biologicals Europe; 1:800), HSP-90α (cat. # 8165, Cell Signaling; 1:1,000), CHI3L1 (cat. # LS-B8213, Life Span BioSciences), APOH (cat. # LS-C314171, Life Span BioSciences, 1:300), tight junction protein 1 (ZO1; cat. # 21773–1-AP, Proteintech; 1:1,000), E-cadherin (cat. # 22018–1-AP, Proteintech; 1:1000), AKT (cat. # 10176–2-AP, Proteintech), phosphorylated (P)-AKT (Ser473; cat. # 4060, Cell Signaling), PI3K (cat. # 2524, Cell Signaling), phosphorylated (P)-PI3K (cat. # 4249T, Cell Signaling), NFκ-B (cat. # 10745–1-AP, Proteintech), C3 (cat. # 21337–1-AP, Proteintech), and β-actin (cat. # abs132184, Absin, Absin Bioscience Co. Ltd.; 1:500).

Techniques:

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Melanoma cell-intrinsic ITGB2 expression and activation by CD44 ( A ) Single-cell (sc) RNA-seq analysis of human ITGB2 gene ( ITGB2 ) expression by patient melanoma (MM) cells versus tumor-infiltrating T cells or endothelial cells (ECs), as depicted by violin plots (median, bold white line; top and bottom quartiles, thin white lines) overlayed with dots representing respective single cells ( B ) Percentages (mean,) of human ITGB2 surface protein expression by patient MM cells, T cells, and ECs ( n = 5 patients) as determined by flow cytometry ( C ) Mean ITGB2 + SOX10 + frequency (%) in benign nevi ( n = 7 patients), primary melanomas ( n = 24 patients), and metastatic melanomas ( n = 13 patients) as determined by multicolor immunofluorescence staining of a patient melanocytic tissue microarray (TMA). Kruskal-Wallis multiple comparisons test was used to assess statistical significance ( D ) Incidence (%) of patient sentinel lymph node (SLN) metastases versus respective primary melanoma biospecimen cohorts ( n = 105) of increasing cancer cell-ITGB2 positivity, 0–2% ( n = 40), 2–25% ( n = 36), >25% ( n = 29), as determined by immunostaining. Frequencies of ITGB2-positive (black bars) and ITGB2-negative (white bars) melanoma cells within each cohort are shown. Fisher’s exact test was performed to determine statistical significance ( E ) Representative multiplex immunofluorescence staining of a patient primary melanoma biopsy for co-expression of ITGB2 (red, all panels) and the melanocytic marker, nuclear SOX-10 (green, first panel), pan T cell marker, CD3 (green, second panel), vascular endothelial marker, CD31 (green, third panel), or macrophage marker, PU.1 (green, fourth panel). Nuclei were counterstained with DAPI (blue). Size bars, 50 μm ( F and G ), Representative immunoblots of ITGB2 protein expression by (F) human melanoma lines, A2058, A375, C8161, FEMX, LOX-IMVI, MDA-MB-435S, and control HSB-2 T lymphoblastic leukemia cells and HUVEC endothelial cells, and (G) murine melanoma lines, B16-F10, YUMM1.7, YUMM3.3, YUMM4.1, YUMM5.2, and control EL-4 T cell lymphoma cells and C166 endothelial cells ( H and I ) Effect of CD44 ab-mediated crosslinking (black bars) versus isotype control ab treatment (white bars) on ITGB2 surface protein expression level (mean fluorescence intensity, MFI, ± SEM) by (H) human and (I) murine melanoma lines and respective cell controls (gray bars) as above, based on FC analysis ( J and K ) Effect of CD44 ab crosslinking as in (H and I) on the activation state of human melanoma cell-ITGB2 as determined by FC (MFI ± SEM) using the activation-sensitive ITGB2 antibody clones (J) KIM-127 and (K) MEM-148. Results are representative of at least n = 3 independent experiments. *, p < 0.05; **, p < 0.01; NS, not significant. See also figs. S1, S2, and S3.

Article Snippet: The following abs and reagents were used for immunohistochemistry and immunofluorescence: unconjugated mouse anti-human ITGB2 ab (clone MEM-48, Novus Biologicals, Cat# NB500-379, RRID: AB_10000712), Dako REAL Detection System, Alkaline Phosphatase/RED (Agilent Dako, Santa Clara, CA, Cat# K5005), Biotin-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Cat# 31800, RRID: AB_228305), AF546-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21123, RRID: AB_2535765), and AF488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21121, RRID: AB_2535764), unconjugated rabbit anti-human SOX10 (clone EPR4007, Abcam, Cat# ab155279, RRID: AB_2650603), unconjugated rabbit anti-human CD3 (clone SP162, Abcam, Cat# ab135372, RRID: AB_2884903), unconjugated rabbit anti-human CD31 (clone EPR3094, Abcam, Cat# ab76533, RRID: AB_1523298), unconjugated rabbit anti-human ICAM-1 (MilliporeSigma, Cat# SAB5700809, RRID: AB_3669069) and Cy3-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A10520, RRID: AB_10563288) or AF488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165), unconjugated mouse anti-human SOX10 (clone 1D2C8, Proteintech, Rosemont, IL, Cat#66786-1-Ig, RRID: AB_2882131) and AF647-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21241, RRID: AB_2535810), unconjugated mouse anti-human PU.1 (clone G148-74, BD Biosciences, Cat# 554268, RRID: AB_395335) and AF488-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21131, RRID: AB_2535771).

Techniques: Expressing, Activation Assay, RNA Sequencing, Flow Cytometry, Multicolor Immunofluorescence Staining, Microarray, Immunostaining, Multiplex Assay, Immunofluorescence, Staining, Marker, Western Blot, Control, Fluorescence, Clone Assay

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Antibody-based blockade of melanoma cell-intrinsic ITGB2 inhibits ICAM-1-dependent adhesion and growth ( A and B ) Relative in vitro adhesion (mean ± SEM) to immobilized ICAM-1 versus negative coating control of (A) human melanoma C8161 and MDA-MB-435S or positive control HSB-2 cells and (B) murine melanoma B16-F10 and YUMM5.2 or positive control EL-4 cells, either untreated (respective left panels) or treated with ITGB2 blocking ab or EDTA pan-integrin antagonist versus isotype control ab (respective right panels). ( C and D ) Tumor growth kinetics in vivo (mean ± SEM) of (C) human C8161 and MDA-MB-435S cells in NSG mice treated with human-specific ITGB2 blocking ab versus isotype control ab or (D) murine B16-F10 and YUMM5.2 cells in NSG mice treated with anti-murine ITGB2 blocking versus isotype control ab. Results in panels (A and B) are representative of and/or pooled from at least n = 3 independent experiments. The unpaired Student’s t test was used to statistically compare two groups and one-way ANOVA with Dunnett’s post-test for comparison of three groups. Panels (C and D) involved n = 5–20 mice per respective treatment group. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth. *, p < 0.05; **, p < 0.01; ***, p < 0.001. See also Figs. and , and , fig. S3

Article Snippet: The following abs and reagents were used for immunohistochemistry and immunofluorescence: unconjugated mouse anti-human ITGB2 ab (clone MEM-48, Novus Biologicals, Cat# NB500-379, RRID: AB_10000712), Dako REAL Detection System, Alkaline Phosphatase/RED (Agilent Dako, Santa Clara, CA, Cat# K5005), Biotin-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Cat# 31800, RRID: AB_228305), AF546-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21123, RRID: AB_2535765), and AF488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21121, RRID: AB_2535764), unconjugated rabbit anti-human SOX10 (clone EPR4007, Abcam, Cat# ab155279, RRID: AB_2650603), unconjugated rabbit anti-human CD3 (clone SP162, Abcam, Cat# ab135372, RRID: AB_2884903), unconjugated rabbit anti-human CD31 (clone EPR3094, Abcam, Cat# ab76533, RRID: AB_1523298), unconjugated rabbit anti-human ICAM-1 (MilliporeSigma, Cat# SAB5700809, RRID: AB_3669069) and Cy3-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A10520, RRID: AB_10563288) or AF488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165), unconjugated mouse anti-human SOX10 (clone 1D2C8, Proteintech, Rosemont, IL, Cat#66786-1-Ig, RRID: AB_2882131) and AF647-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21241, RRID: AB_2535810), unconjugated mouse anti-human PU.1 (clone G148-74, BD Biosciences, Cat# 554268, RRID: AB_395335) and AF488-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21131, RRID: AB_2535771).

Techniques: In Vitro, Control, Positive Control, Blocking Assay, In Vivo, Comparison

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Antibody-based ITGB2 blockade or host Icam1 deficiency inhibit melanoma metastasis ( A to C ) Effect of anti-murine ITGB2 blocking ab versus isotype control ab on tumorigenesis of B16-F10 and YUMM5.2 cells in wildtype (WT) C57BL/6 mice. (A) Tumor growth kinetics (mean ± SEM), (B) relative intratumoral T cell levels, and (C) relative lung metastasis of GFP-expressing melanoma cells were determined by qPCR-based quantitation of genomic Cd3 or GFP in tumor and lung tissue, respectively. (B ) Primer specificity for Cd3 was validated using positive control murine T cells and negative control B16-F10 and YUMM5.2 cells. (C) Specificity of GFP primers was authenticated using positive control GFP-expressing B16-F10 and YUMM5.2 cells and negative control lungs obtained from WT mice without tumors. ( D to F ) Effect of anti-murine ITGB2 blocking ab versus isotype control ab on tumorigenesis of B16-F10 and YUMM5.2 cells in Icam1 −/− C57BL/6 mice. (D) Tumor growth kinetics (mean ± SEM), (E) intratumoral T cell levels, and (F) lung metastasis in Icam1- deficient mice were determined by qPCR analysis using positive and negative cell and sample controls, as above. Panels (A and D) involved n = 16–20 mice per respective treatment group. Results in panels (B, C, E, and F) are representative of and/or pooled from at least n = 3 independent experiments. Tumor control groups in panels B and E, C and F are identical, respectively. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth in panels (A and D). Data in (B, C, E, and F) were statistically compared using the unpaired Student’s t test. *, p < 0.05; NS, not significant; nd, not detected. See also Figs. and , fig. S3

Article Snippet: The following abs and reagents were used for immunohistochemistry and immunofluorescence: unconjugated mouse anti-human ITGB2 ab (clone MEM-48, Novus Biologicals, Cat# NB500-379, RRID: AB_10000712), Dako REAL Detection System, Alkaline Phosphatase/RED (Agilent Dako, Santa Clara, CA, Cat# K5005), Biotin-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Cat# 31800, RRID: AB_228305), AF546-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21123, RRID: AB_2535765), and AF488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21121, RRID: AB_2535764), unconjugated rabbit anti-human SOX10 (clone EPR4007, Abcam, Cat# ab155279, RRID: AB_2650603), unconjugated rabbit anti-human CD3 (clone SP162, Abcam, Cat# ab135372, RRID: AB_2884903), unconjugated rabbit anti-human CD31 (clone EPR3094, Abcam, Cat# ab76533, RRID: AB_1523298), unconjugated rabbit anti-human ICAM-1 (MilliporeSigma, Cat# SAB5700809, RRID: AB_3669069) and Cy3-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A10520, RRID: AB_10563288) or AF488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165), unconjugated mouse anti-human SOX10 (clone 1D2C8, Proteintech, Rosemont, IL, Cat#66786-1-Ig, RRID: AB_2882131) and AF647-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21241, RRID: AB_2535810), unconjugated mouse anti-human PU.1 (clone G148-74, BD Biosciences, Cat# 554268, RRID: AB_395335) and AF488-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21131, RRID: AB_2535771).

Techniques: Blocking Assay, Control, Expressing, Quantitation Assay, Positive Control, Negative Control

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: CRISPR/Cas9-based genetic knockout of melanoma cell-intrinsic Itgb2 suppresses adhesion to ICAM-1 and resultant tumor growth ( A ) Validation of CRISPR/Cas9-mediated stable KO of Itgb2 gene and ITGB2 protein in B16-F10 and YUMM5.2 melanoma cells as determined by RT-qPCR (left panel) and immunoblotting (right panel). ( B to F ) Itgb2 KO versus respective Cas9 control B16-F10 and YUMM5.2 tumor cell relative (B) in vitro adhesion (mean ± SEM) to immobilized ICAM-1, with or without negative control EDTA treatment, (C) in vitro growth (mean ± SEM) as determined by CellTiter-Glo-based luminescence analysis, and (D to F) in vivo tumor growth kinetics (mean ± SEM) in (D) NSG mice, (E) C57BL/6 mice, and (F) Icam1 −/− C57BL/6 mice. ( G ) Relative Icam1 gene expression in B16-F10 and YUMM5.2 tumors from C57BL/6 mice (black bars) versus Icam1 −/− C57BL/6 mice (white bars), with positive control murine T cells and C166 endothelial cells shown (gray bars). ( H ) scRNA-seq analysis of human ICAM1 gene expression in patient melanoma (MM) cells, tumor-infiltrating T cells, and endothelial cells (ECs) as depicted by violin plots (median, bold white line; top and bottom quartiles, thin white lines) overlayed with dots representing respective single cells. ( I ) Percentages (mean) of human ICAM-1 surface protein expression by patient MM cells, T cells, and ECs ( n = 5 patients) as determined by FC. ( J ) Multiplex immunofluorescence staining of a representative ( n = 4 patients) clinical melanoma biospecimen for expression of the melanocytic marker, nuclear SOX-10 (red, first panel), ITGB2 (yellow, second panel), and ICAM-1 (green, third panel). The merged image is also shown (fourth panel). Nuclei were counterstained with DAPI (blue). Size bars, 50 μm. Results in panels (A, B, C, and G) are representative of and/or pooled from at least n = 3 independent experiments. The unpaired Student’s t test was used to statistically compare two groups and one-way ANOVA with Dunnett’s post-test for comparison of three groups. Panels (D to F) involved n = 10 mice per respective melanoma cell variant. Repeated-measures two-way ANOVA was used to assess statistical differences in tumor growth. **, p < 0.01; ***, p < 0.001; NS, not significant; nd, not detected. See also Figs. and 4, figs. S3 and S4

Article Snippet: The following abs and reagents were used for immunohistochemistry and immunofluorescence: unconjugated mouse anti-human ITGB2 ab (clone MEM-48, Novus Biologicals, Cat# NB500-379, RRID: AB_10000712), Dako REAL Detection System, Alkaline Phosphatase/RED (Agilent Dako, Santa Clara, CA, Cat# K5005), Biotin-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Cat# 31800, RRID: AB_228305), AF546-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21123, RRID: AB_2535765), and AF488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21121, RRID: AB_2535764), unconjugated rabbit anti-human SOX10 (clone EPR4007, Abcam, Cat# ab155279, RRID: AB_2650603), unconjugated rabbit anti-human CD3 (clone SP162, Abcam, Cat# ab135372, RRID: AB_2884903), unconjugated rabbit anti-human CD31 (clone EPR3094, Abcam, Cat# ab76533, RRID: AB_1523298), unconjugated rabbit anti-human ICAM-1 (MilliporeSigma, Cat# SAB5700809, RRID: AB_3669069) and Cy3-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A10520, RRID: AB_10563288) or AF488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165), unconjugated mouse anti-human SOX10 (clone 1D2C8, Proteintech, Rosemont, IL, Cat#66786-1-Ig, RRID: AB_2882131) and AF647-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21241, RRID: AB_2535810), unconjugated mouse anti-human PU.1 (clone G148-74, BD Biosciences, Cat# 554268, RRID: AB_395335) and AF488-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21131, RRID: AB_2535771).

Techniques: CRISPR, Knock-Out, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Control, In Vitro, Negative Control, In Vivo, Gene Expression, Positive Control, Expressing, Multiplex Assay, Immunofluorescence, Staining, Marker, Comparison, Variant Assay

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: The melanoma cell-ITGB2:ICAM-1 axis stimulates downstream Wnt pathway activation, the inhibition of which suppresses cancer cell:ICAM-1 adhesion ( A ) Heatmaps of differentially expressed genes (DEGs) exhibiting pathway interconnectivity ( n = 51) in Itgb2 KO versus control YUMM5.2 tumors and which showed consistent trends in both NSG (left panel) and wildtype (WT) C57BL/6 mice (middle panel), but not in Icam1 −/− C57BL/6 hosts (right panel), as determined by RNA-seq analysis. ( B ) Protein-protein interaction and cluster map (STRING) of 22 of the 51 DEGs described in (A) exhibiting the strongest interaction scores. Respective network clusters (gray ovals) and relative strengths of direct protein-protein interactions (stronger, wider lines; weaker, thinner lines) as well as indirect associations (dashed lines) are shown. Proteins without any designated cluster associations were omitted. The paired Wilcoxon test was used to assess statistical significance. ( C ) Magnitude of difference in expression of each Wnt pathway DEG in Itgb2 KO versus Cas9 control melanomas (log fold change) as in (A) and identified in the Gene Ontology Biological Process (GOBP) database. Wnt signaling effectors were grouped into activating ( Frat2 , Kpna1 , Wnt5a , Wnt5b ) versus inhibitory ( Dkk2 , Igfbp4 , Kank1 , Notum ) cohorts. Medians are represented by horizontal bars in box and whiskers plots. ( D ) Validation by RT-qPCR (fold change) of Wnt effector DEGs as in (C) using independent Itgb2 KO versus Cas9 control YUMM5.2 tumor biospecimens from NSG, WT, or Icam1 −/− C57BL/6 mice. Medians are represented by horizontal bars in box and whiskers plots. ( E ) Representative immunoblots of canonical Wnt mediators, active (non-p) β-catenin and LEF-1, and ACTB loading control (left), and non-canonical Wnt effector, p-VANGL2, and respective total controls (right) in Itgb2 KO versus Cas9 control YUMM5.2 melanoma cells. ( F ) Representatie immunoblots of Wnt signaling mediators as in (E) of YUMM5.2 melanoma cells treated with the Wnt inhibitors, pyrvinium pamoate, LGK974, or zamaporvint, versus vehicle control. ( G and H ) Relative in vitro adhesion (mean ± SEM) to immobilized ICAM-1 as determined by CellTiter-Glo-based luminescence analysis of (G) Itgb2 KO versus Cas9 control YUMM5.2 variants and (H) anti-murine ITGB2 blocking ab versus isotype control ab treated YUMM5.2 wildtype cells, in the combined presence or absence of pyrvinium pamoate, LGK974, zamaporvint, or vehicle control. The paired Student’s t test was used to assess statistical significance. Panels (A, B, C, and D) are representative of n = 2–6 tumors per variant group in each respective animal host. Results in (E, F, G, and H) are representative of and/or pooled from at least n = 2–7 independent experiments each. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. See also Figs. and , and , figs. S5 and S6

Article Snippet: The following abs and reagents were used for immunohistochemistry and immunofluorescence: unconjugated mouse anti-human ITGB2 ab (clone MEM-48, Novus Biologicals, Cat# NB500-379, RRID: AB_10000712), Dako REAL Detection System, Alkaline Phosphatase/RED (Agilent Dako, Santa Clara, CA, Cat# K5005), Biotin-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Cat# 31800, RRID: AB_228305), AF546-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21123, RRID: AB_2535765), and AF488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21121, RRID: AB_2535764), unconjugated rabbit anti-human SOX10 (clone EPR4007, Abcam, Cat# ab155279, RRID: AB_2650603), unconjugated rabbit anti-human CD3 (clone SP162, Abcam, Cat# ab135372, RRID: AB_2884903), unconjugated rabbit anti-human CD31 (clone EPR3094, Abcam, Cat# ab76533, RRID: AB_1523298), unconjugated rabbit anti-human ICAM-1 (MilliporeSigma, Cat# SAB5700809, RRID: AB_3669069) and Cy3-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A10520, RRID: AB_10563288) or AF488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165), unconjugated mouse anti-human SOX10 (clone 1D2C8, Proteintech, Rosemont, IL, Cat#66786-1-Ig, RRID: AB_2882131) and AF647-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21241, RRID: AB_2535810), unconjugated mouse anti-human PU.1 (clone G148-74, BD Biosciences, Cat# 554268, RRID: AB_395335) and AF488-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21131, RRID: AB_2535771).

Techniques: Activation Assay, Inhibition, Control, RNA Sequencing, Protein-Protein interactions, Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot, In Vitro, Blocking Assay, Variant Assay

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Wnt antagonism suppresses ITGB2:ICAM-1-dependent melanoma growth in vivo ( A and B ) Tumor growth kinetics (mean ± SEM) of (A) Itgb2 KO versus Cas9 control YUMM5.2 variant cells or (B) YUMM5.2 wildtype cells treated with anti-murine ITGB2 blocking ab versus isotype control ab, with or without concurrent administration of the Wnt inhibitors, pyrvinium pamoate, LGK974, zamaporvint, as well as vehicle control in NSG (left panel), wildtype (WT) C57BL/6 (middle panel), or Icam1 −/− C57BL/6 mice (right panel). Because tumorigenicity experiments evaluating LGK974 and zamaporvint effects were conducted concurrently, vehicle control groups for both drugs are identical. Panels (A and B) involved n = 6–10 mice per respective treatment group. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth in panels. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. See also Fig.

Article Snippet: The following abs and reagents were used for immunohistochemistry and immunofluorescence: unconjugated mouse anti-human ITGB2 ab (clone MEM-48, Novus Biologicals, Cat# NB500-379, RRID: AB_10000712), Dako REAL Detection System, Alkaline Phosphatase/RED (Agilent Dako, Santa Clara, CA, Cat# K5005), Biotin-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Cat# 31800, RRID: AB_228305), AF546-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21123, RRID: AB_2535765), and AF488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21121, RRID: AB_2535764), unconjugated rabbit anti-human SOX10 (clone EPR4007, Abcam, Cat# ab155279, RRID: AB_2650603), unconjugated rabbit anti-human CD3 (clone SP162, Abcam, Cat# ab135372, RRID: AB_2884903), unconjugated rabbit anti-human CD31 (clone EPR3094, Abcam, Cat# ab76533, RRID: AB_1523298), unconjugated rabbit anti-human ICAM-1 (MilliporeSigma, Cat# SAB5700809, RRID: AB_3669069) and Cy3-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A10520, RRID: AB_10563288) or AF488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165), unconjugated mouse anti-human SOX10 (clone 1D2C8, Proteintech, Rosemont, IL, Cat#66786-1-Ig, RRID: AB_2882131) and AF647-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21241, RRID: AB_2535810), unconjugated mouse anti-human PU.1 (clone G148-74, BD Biosciences, Cat# 554268, RRID: AB_395335) and AF488-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21131, RRID: AB_2535771).

Techniques: In Vivo, Control, Variant Assay, Blocking Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Round window membrane extracellular vesicles facilitate inner ear drug delivery

doi: 10.1016/j.jconrel.2025.114153

Figure Lengend Snippet: RWM cells release EVs. (A) A schematic showing the extracellular vesicles (EVs) that are released from cells and can be loaded with proteins, mRNAs, lipids, small molecules, and more. Three groups of vesicle sources are shown schematically: MSC EVs: released by porcine bone marrow stem cells, Epi/Fibro EVs: released by epithelial and fibroblast cells of porcine round window membrane-RWM, and Liposomes. The RWM is the port of entry to the inner ear and consists of an outer epithelial layer, a middle fibroblast layer, and an inner epithelial layer. (B) The nanoparticle tracking shows the size distribution of the nanovesicles released by RWM Epithelial (Epi) and Fibroblast (Fibro) cells, as well as Mesenchymal stem cell (MSC), before and after loading with red fluorescent protein (RFP). The transmission electron microscopy (TEM) micrographs showing all three vesicles before and after loading confirm the integrity of the nanovesicles after loading. (C) The flow cytometry analysis of CD63 antibody at FITC-A channel for Epi, Fibro, and MSC vesicles confirmed the CD63+ nanovesicles. The Ctrl group contains only the secondary antibody. (D) The immunoTEM micrographs of the RWM EVs against gold-conjugated CD9, CD63, and CD81 (exosome markers) confirm exosome identity of EVs derived from RWM Fibroblast Cells via Heat Shock. (E) The western blotting analysis of epithelial and fibroblast EVs isolated by serum deprivation (Epi, Fibro) or heat shock (Epi-HS, Fibro-HS) using CD9, CD63, and CD81 antibodies further confirms the nature of nanovesicles as EVs. PNGase F was used to analyze whether a protein is N-glycosylated and to study the impact of glycosylation on its molecular weight. In PNGase + samples, the band between 50 and 90 KDa disappears, and a new band between 30 and 38 KDa is present, confirming the glycosylation of the CD9, CD63, and CD81 proteins.

Article Snippet: Selected antibodies were CD9 (NB500-327, Novus Biologicals), CD63 (NBP2-42225, Novus Biologicals), CD81 (NB100-65805, Novus Biologicals), and B-Actin (3700, Cell Signaling Technologies).

Techniques: Membrane, Liposomes, Transmission Assay, Electron Microscopy, Flow Cytometry, Derivative Assay, Western Blot, Isolation, Glycoproteomics, Molecular Weight

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Round window membrane extracellular vesicles facilitate inner ear drug delivery

doi: 10.1016/j.jconrel.2025.114153

Figure Lengend Snippet: Loading in RWM EVs leads to higher passage across RWM ex vivo and in vivo in pigs. (A) The schematic of the ex-vivo and in-vivo transport test is shown. For the ex-vivo method, the substances are placed on top of the intact, excised RWM, as previously described , in a transwell chamber (without mesh). For the in-vivo method, substances were delivered via IT injection into the middle ear, as previously described , and the inner ear perilymph (20 μL) was collected 1 h after injection from the RWM via a microcapillary tube. The perilymph was then analyzed via mass spectrometry. (B) The concentration of dexamethasone fluorescein (DexF) after passage across RWM explants in transwell and the permeability (Kp) of RWM explant for DexF are shown when DexF is loaded inside Fibro HSEVs, Lipo, and MSC EVs. The Fibro HSEVs had significantly higher passage ex-vivo than naked DexF (biological replicates n: 3, nested 1-way ANOVA p -value: 0.0019). The Fibro HSEVs significantly enhanced the RWM permeability for DexF ex vivo vs naked DexF (biological replicates n: 3, nested 1-way ANOVA p-value: 0.0380). Lipo and MSC EVs did not significantly enhance the RWM permeability for DexF (biological replicates n:3, nested 1-way ANOVA). A plate reader was used for DexF concentration analysis. (C) ImmunoTEM micrographs of the RWM tissue after the Fibro HS EVs passage show the presence of EVs (gold-conjugated CD9, CD63, and CD81) in the middle layer of RWM, confirming their passage across the epithelial barrier. The B-actin used as a control shows specific staining within the fibroblast cells of the RWM. The top row shows lower magnifications, and the bottom row shows higher magnifications. (D) No difference was observed for the concentration of dexamethasone sodium phosphate (DSP) between DSP alone and DSP-loaded Fibro HSEVs ex vivo and in vivo, or between DSP-loaded Fibro HS EVs ex vivo as compared to Fibro HSEVs in vivo. (biological replicates n: 5, One way ANOVA; p -values: 0.9999, 0.1343, 0.8779 for DSP vs. EVs-DSP ex vivo, DSP vs. EVs-DSP in vivo, and EVs-DSP ex vivo vs. EVs-DSP in vivo, respectively. The permeability of the RWM for DSP significantly increased when DSP is loaded inside Fibro HSEVs, both ex vivo and in vivo (biological replicates n: 5, One way ANOVA; p-values:0.0234 and 0.0265 for DSP vs EVs-DSP in ex vivo and in vivo, respectively). No change was observed for EVs-DSP ex vivo compared to in vivo (biological replicates n: 5, One way ANOVA; p-value 0.0693 for EVs-DSP ex vivo vs in vivo). Mass spectrometry was used for DSP concentration analysis.

Article Snippet: Selected antibodies were CD9 (NB500-327, Novus Biologicals), CD63 (NBP2-42225, Novus Biologicals), CD81 (NB100-65805, Novus Biologicals), and B-Actin (3700, Cell Signaling Technologies).

Techniques: Ex Vivo, In Vivo, Injection, Mass Spectrometry, Concentration Assay, Permeability, Control, Staining