Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) Immunoprecipitation followed by mass spectrometry of SIRPα-associated proteins. ( A ) Schematic representation of assay. ( B ) Plasma membrane-associated proteins found in SIRPα immunoprecipitates from WT BMDMs, but not from SIRPα KO BMDMs. c , Co-immunoprecipitation assay of SIRPα, CD18 and CD11b in WT and SIRPα KO BMDMs. IP, immunoprecipitation. Abs, antibodies. ( D to F ) FRET assays. ( D ) Schematic representation of FRET assay in HEK293T cells. ( E and F ) Representative confocal microscopy images ( E ) and compiled data ( F ) of FRET assays with donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of control (Ctrl) IgG, CD18 mAb GAME-46 or CD11b mAb 5C6. Yellow to purple spectrum denotes strong to weak FRET. DIC, differential interference contrast. Scale bars, 5 μm. ( G and H ) LUV-FRET assay. ( G ) Schematic representation of LUV-FRET assay. ( H ), Time-course of donor-labeled SIRPα fluorescence intensity after addition of acceptor-labeled CD18 or CD11b, monitored with a real-time plate reader. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( C , E and H ) are representative of 3 independent experiments. Results in ( B and F ) are pooled from a total of 3 independent experiments. Each symbol in ( F ) represents one cell.

Article Snippet: Mouse CD18 mAb M18/2 was purchased from Developmental Studies Hybridoma Bank (Iowa City, IA).

Techniques: Immunoprecipitation, Mass Spectrometry, Clinical Proteomics, Membrane, Co-Immunoprecipitation Assay, Confocal Microscopy, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) FRET assays with SIRPα and SIRPβ1a. ( A ) A schematic representation of SIRPα and SIRPβ1a, with their 1 IgV domain and 2 IgC domains, is depicted. ( B ) Compiled data of 3 independent experiments using donor-labeled SIRPα or SIRPβ1a, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( C and D ) FRET assays using SIRPα IgV domain. ( C ) A schematic representation of a SIRPα variant having only the IgV domain is shown. ( D ) Compiled data of 3 independent experiments using donor-labeled SIRPα IgV, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( E - H ) FRET assays using SIRPα variants carrying non-conserved residues from SIRPβ1a. ( E and G ) Schematic representations of SIRPα variants. ( F and H ) Compiled data from 3 independent experiments using donor-labeled SIRPα variants, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( I to K ) Proximity ligation assay (PLA) of SIRPα and CD18 in BMDMs expressing or not the indicated SIRPα variants. (I) Flow cytometry analyses of SIRPα expression. ( J and K ) Representative confocal microscopy images ( J ) and compiled data from 3 independent experiments ( K ) of PLA for SIRPα and CD18. Scale bar, 10 μm. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( I and J ) are representative of 3 independent experiments. Results in ( B , D , F , H and K ) are pooled from 3 independent experiments. Each symbol in ( B , D , F , H and K ) represents one cell or mouse.

Article Snippet: Mouse CD18 mAb M18/2 was purchased from Developmental Studies Hybridoma Bank (Iowa City, IA).

Techniques: Labeling, Variant Assay, Proximity Ligation Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: Mouse CD18 mAb M18/2 was purchased from Developmental Studies Hybridoma Bank (Iowa City, IA).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: Mouse CD18 mAb M18/2 was purchased from Developmental Studies Hybridoma Bank (Iowa City, IA).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis

Journal: MethodsX

Article Title: Feline leukocyte immunophenotyping: an optimised whole-blood flow cytometry protocol

doi: 10.1016/j.mex.2026.103869

Figure Lengend Snippet: Representative leukocyte-gated histograms illustrating antibody titration for feline leukocyte immunophenotyping. All histograms display singlet leukocytes, defined by FSC-A versus SSC-A morphological gating, followed by FSC—H versus FSC-A singlet discrimination. Titration was performed for CD18, CD21, CD45R, CD4, CD5 and CD8 monoclonal antibodies using three antibody volumes: 10 µL (1:10 dilution), 5.0 µL (1:20 dilution), 3.0 µL (1:33 dilution) and 1.5 µL (1:66 dilution). Minimal working volumes were selected based on optimal signal-to-noise ratios.

Article Snippet: Step 2 – Leukocytes extracellular staining Materials • EDTA Whole blood sample ± Transfix® • 200 μl pipettes • 100 μl pipettes • 10 μl pipettes • Flow cytometry tubes • Permanent marker • Cytometer tube rack Reagents • Monoclonal antibodies: ○ CD5 Anti-cat – clone FE1.1B11 (BIO-RAD®) ○ CD4 Anti-cat – clone vpg34 (BIO-RAD®) ○ CD8 Anti-cat alpha/beta purified – clone vpg9 (BIO-RAD®) ○ Rat Anti-Mouse IgG1 – clone X56 (BIO-RAD®) ○ CD18 Mouse Anti-Dog – clone CA1.4E9 (BIO-RAD®) ○ CD21 Mouse Anti-Dog – clone CA2.1D6 (BIO-RAD®) ○ CD45R Rat Anti-Mouse – clone RA3–6B2 (BIO-RAD®) • 10x Red blood cells (RBC) lysis buffer solution (BD FACSTM lysing solution) • PBS 1% solution Equipment • Countess TM 3 (Thermo Fisher Scientific, USA) • Freezer • Dark incubation chamber • Timer • Vortex (MX-S®, China) • Centrifuge (model 5810R, Eppendorf®, Germany) • Flow cytometry analyser BD FACSCanto II (Becton Dickinson (BD), San Jose, USA) Methods 1.

Techniques: Titration, Bioprocessing

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