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Functional heterologous receptor assay involving CHO-K1 cells expressing the A. aegypti CAPA receptor to determine CAPA titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13– 10 −5 mol l −1 doses of Aedes CAPA-1 peptide (EC 50 =1.83 nmol l −1 ). Luminescence was normalized to <t>BSA</t> <t>medium</t> and plotted relative to the maximal response (10 −5 mol l −1 ); data represent the means±s.e.m. ( n =4). (B) Measured concentrations of CAPA peptide levels in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed). Significantly different CAPA peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post-hoc test (* P <0.05); data represent means±s.e.m. ( n =8–11).

Bsa Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Dynamics of release and activity of select neuropeptides post-bloodmeal in the female mosquito, Aedes aegypti"

Article Title: Dynamics of release and activity of select neuropeptides post-bloodmeal in the female mosquito, Aedes aegypti

Journal: The Journal of Experimental Biology

doi: 10.1242/jeb.250150

Figure Legend Snippet: Functional heterologous receptor assay involving CHO-K1 cells expressing the A. aegypti CAPA receptor to determine CAPA titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13– 10 −5 mol l −1 doses of Aedes CAPA-1 peptide (EC 50 =1.83 nmol l −1 ). Luminescence was normalized to BSA medium and plotted relative to the maximal response (10 −5 mol l −1 ); data represent the means±s.e.m. ( n =4). (B) Measured concentrations of CAPA peptide levels in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed). Significantly different CAPA peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post-hoc test (* P <0.05); data represent means±s.e.m. ( n =8–11).

Techniques Used: Functional Assay, Expressing, Comparison

Functional heterologous receptor assay of CHO-K1 cells expressing the A. aegypti kinin receptor to determine Aedae kinin titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13 to 10 −5 mol l −1 doses of CDP (EC 50 =11.47 nmol l −1 ) and leucokinin (LK) (EC 50 =4.34 nmol l −1 ). (B) Raw luminescent response following addition of 10 −6 mol l −1 dose of representative neuropeptides belonging to several insect peptide families; different letters denote bars that are significantly different from control (BSA assay medium) as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test ( P <0.0001). For peptide sequence information, see <xref ref-type=

Table 2 . Individual data points ( n =3) from each experimental replicate are shown. (C) Concentration of kinin peptides in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed; relative to LK standard curve). Significantly different kinin peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test (** P <0.001); data represent means±s.e.m. ( n =10–15). " title="... denote bars that are significantly different from control (BSA assay medium) as determined by a one-way ANOVA ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Functional heterologous receptor assay of CHO-K1 cells expressing the A. aegypti kinin receptor to determine Aedae kinin titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13 to 10 −5 mol l −1 doses of CDP (EC 50 =11.47 nmol l −1 ) and leucokinin (LK) (EC 50 =4.34 nmol l −1 ). (B) Raw luminescent response following addition of 10 −6 mol l −1 dose of representative neuropeptides belonging to several insect peptide families; different letters denote bars that are significantly different from control (BSA assay medium) as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test ( P <0.0001). For peptide sequence information, see Table 2 . Individual data points ( n =3) from each experimental replicate are shown. (C) Concentration of kinin peptides in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed; relative to LK standard curve). Significantly different kinin peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test (** P <0.001); data represent means±s.e.m. ( n =10–15).

Techniques Used: Functional Assay, Expressing, Control, Comparison, Sequencing, Concentration Assay

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Journal: The Journal of Experimental Biology

Article Title: Dynamics of release and activity of select neuropeptides post-bloodmeal in the female mosquito, Aedes aegypti

doi: 10.1242/jeb.250150

Figure Lengend Snippet: Functional heterologous receptor assay involving CHO-K1 cells expressing the A. aegypti CAPA receptor to determine CAPA titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13– 10 −5 mol l −1 doses of Aedes CAPA-1 peptide (EC 50 =1.83 nmol l −1 ). Luminescence was normalized to BSA medium and plotted relative to the maximal response (10 −5 mol l −1 ); data represent the means±s.e.m. ( n =4). (B) Measured concentrations of CAPA peptide levels in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed). Significantly different CAPA peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post-hoc test (* P <0.05); data represent means±s.e.m. ( n =8–11).

Article Snippet: Approximately 48 h post-transfection, cells were dislodged from the culture flasks using 5 mmol l −1 ethylenediaminetetraacetic acid (EDTA; Life Technologies, Burlington, ON, Canada) in DPBS and cells were resuspended in BSA medium (DMEM-F12 medium containing 0.1% bovine serum albumin, 1X antimycotic-antibiotic) at a concentration of 10 6 –10 7 cells ml −1 quantified using a Countess II FL cell counter (Thermo Fisher Scientific, Burlington, ON, Canada).

Techniques: Functional Assay, Expressing, Comparison

Journal: The Journal of Experimental Biology

Article Title: Dynamics of release and activity of select neuropeptides post-bloodmeal in the female mosquito, Aedes aegypti

doi: 10.1242/jeb.250150

Figure Lengend Snippet: Functional heterologous receptor assay of CHO-K1 cells expressing the A. aegypti kinin receptor to determine Aedae kinin titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13 to 10 −5 mol l −1 doses of CDP (EC 50 =11.47 nmol l −1 ) and leucokinin (LK) (EC 50 =4.34 nmol l −1 ). (B) Raw luminescent response following addition of 10 −6 mol l −1 dose of representative neuropeptides belonging to several insect peptide families; different letters denote bars that are significantly different from control (BSA assay medium) as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test ( P <0.0001). For peptide sequence information, see Table 2 . Individual data points ( n =3) from each experimental replicate are shown. (C) Concentration of kinin peptides in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed; relative to LK standard curve). Significantly different kinin peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test (** P <0.001); data represent means±s.e.m. ( n =10–15).

Techniques: Functional Assay, Expressing, Control, Comparison, Sequencing, Concentration Assay

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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

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.

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

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.

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.

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

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