icam1 primary antibodies  (Proteintech)

Journal: Frontiers in Oncology

Article Title: The impact of macrophages on endothelial cells is potentiated by cycling hypoxia: Enhanced tumor inflammation and metastasis

doi: 10.3389/fonc.2022.961753

Figure Lengend Snippet: ICAM1 mRNA expression in EA.hy926 cells and HUVECs incubated with macrophage-conditioned media. THP-1–derived unpolarized, M1-like, and M2-like macrophages were exposed to normoxia (N), chronic hypoxia (chH), or cycling hypoxia (cyH) for 6 h and were then left for 16 h in normoxic air to produce macrophage-conditioned media. Thereafter, EA.hy926 cells (A) and HUVECs (B) were incubated for 24 h with macrophage-conditioned media. mRNA expression of ICAM1 in EA.hy926 cells (A) and HUVECs (B) was assessed by RT-qPCR (n = 4, mean ± 1 SEM). Ctrl 1 corresponds to endothelial cells incubated with CO 2 -independent medium. Ctrl 2 corresponds to endothelial cells incubated with EGM-2 medium. Statistical analysis was performed using Student’s t test. *p < 0.05; **p < 0.01.

Article Snippet: Cells were washed three times in PBS and were blocked in PBS containing 2% of bovine serum albumin (BSA) for 30 min. Then, cells were incubated overnight (O/N) at 4°C with anti-ICAM1 primary antibody (diluted 1:60 in 2% BSA in PBS; #BBA3, R&D Systems).

Techniques: Expressing, Incubation, Derivative Assay, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: The impact of macrophages on endothelial cells is potentiated by cycling hypoxia: Enhanced tumor inflammation and metastasis

doi: 10.3389/fonc.2022.961753

Figure Lengend Snippet: ICAM1 protein abundance in EA.hy926 cells and HUVECs incubated with macrophage-conditioned media. THP-1–derived unpolarized, M1-like, and M2-like macrophages were exposed to normoxia (N), chronic hypoxia (chH), or cycling hypoxia (cyH) for 6 h and were then left for 16 h in normoxic air to produce macrophage-conditioned media. Thereafter, EA.hy926 cells and HUVECs were incubated for 48 h with macrophage-conditioned media. ICAM1 protein abundance in EA.hy926 cells (A) and HUVECs (B) was analyzed by immunofluorescence (n = 2). ICAM1 protein abundance in EA.hy926 cells (C) was analyzed by western blot (n = 3, mean ± 1 SEM). Ctrl 1 corresponds to endothelial cells incubated with CO 2 -independent medium. Ctrl 2 corresponds to endothelial cells incubated with DHGL-1 (A) or EGM-2 (B) . Incubation of endothelial cells with TNFα (1 ng/ml) for 16 h was used as positive control. Statistical analysis was performed using Student’s t test. *p < 0.05; **p < 0.01.

Article Snippet: Cells were washed three times in PBS and were blocked in PBS containing 2% of bovine serum albumin (BSA) for 30 min. Then, cells were incubated overnight (O/N) at 4°C with anti-ICAM1 primary antibody (diluted 1:60 in 2% BSA in PBS; #BBA3, R&D Systems).

Techniques: Quantitative Proteomics, Incubation, Derivative Assay, Immunofluorescence, Western Blot, Positive Control

Journal: Journal of Nanobiotechnology

Article Title: New insights into allergic rhinitis treatment: MSC nanovesicles targeting dendritic cells

doi: 10.1186/s12951-024-02748-2

Figure Lengend Snippet: Influence of P-D2-EVs on Fut1-mediated fucosylation of ICAM1. A Western blot analysis of UEA-1-enriched mDCs under different treatments. B Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. C IP-based detection and quantification of ICAM1 binding to UEA1 in each group. D Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm. E Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. F IP-based detection and quantification of ICAM1 binding to UEA1 in each group. G Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm. H Schematic representation of Fut1-OE-iDCs treated with 2DGal; (I) Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. J IP-based detection and quantification of ICAM1 binding to UEA1 in each group. K Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm; UEA1: Ulex europaeus agglutinin 1, 2DGal: 2-deoxy- d -galactose. * indicates statistical significance compared to the control group or between two groups, with P < 0.05; all experiments were repeated three times

Article Snippet: Slides were incubated overnight with primary antibodies against ICAM1 (Thermofisher, MA5-43106) at a dilution of 1:60.

Techniques: Western Blot, Binding Assay, Immunofluorescence, Staining, Control

Journal: Journal of Nanobiotechnology

Article Title: New insights into allergic rhinitis treatment: MSC nanovesicles targeting dendritic cells

doi: 10.1186/s12951-024-02748-2

Figure Lengend Snippet: Influence of P-D2-EVs on DC metabolism via the Fut1/ICAM1/P38 MAPK pathway. A , B Quantification of p-P38 and P38 expression, as well as the p-P38/P38 ratio, in mDCs from different treatment groups using Western blot. C mRNA expression of IL10 in mDCs from different treatment groups measured by RT-qPCR. D Levels of IL10 in the supernatant of mDCs from different treatment groups analyzed using ELISA. E Analysis of intracellular IL-10 levels in mDCs from different treatment groups using flow cytometry. F Schematic representation of the culture conditions for mDCs, mDCs + 2DGal, and m + 2DGal + Anisomycin groups. G Quantification of p-P38 and P38 expression, as well as the p-P38/P38 ratio, in mDCs from different treatment groups using Western blot. H mRNA expression of IL10 in mDCs from different treatment groups measured by RT-qPCR. I Levels of IL10 in the supernatant of mDCs from different treatment groups analyzed using ELISA. J Analysis of intracellular IL-10 levels in mDCs from different treatment groups using flow cytometry; 2DGal: 2-deoxy- d -galactose; *indicates statistical significance compared to the control group or between two groups, with P < 0.05; all experiments were repeated three times

Article Snippet: Slides were incubated overnight with primary antibodies against ICAM1 (Thermofisher, MA5-43106) at a dilution of 1:60.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control

Journal: Journal of Nanobiotechnology

Article Title: New insights into allergic rhinitis treatment: MSC nanovesicles targeting dendritic cells

doi: 10.1186/s12951-024-02748-2

Figure Lengend Snippet: The impact of P-D2-EVs on regulating the Fut1/ICAM1/P38 MAPK pathway and IL10 metabolism in DCs on Th2 differentiation. A Schematic representation of co-culturing mDCs with CD4 + T cells. B Flow cytometric analysis of the percentage of IL4 and GATA3 in mDCs co-cultured with CD4 + T cells under different treatments. C RT-qPCR analysis of mRNA expression levels of IL4 and GATA3 in mDCs co-cultured with CD4 + T cells under different treatments. D ELISA analysis of IL4 levels in the supernatant of mDCs co-cultured with CD4 + T cells under different treatments. E Flow cytometric analysis of the percentage of IL4 and GATA3 in mDCs co-cultured with CD4 + T cells under different treatments. F RT-qPCR analysis of mRNA expression levels of IL4 and GATA3 in mDCs co-cultured with CD4 + T cells under different treatments. G ELISA analysis of IL4 levels in the supernatant of mDCs co-cultured with CD4 + T cells under different treatments; * indicates statistical significance compared to the control group or between two groups (P < 0.05); all experiments repeated three times. H Schematic diagram of co-culture of CD4 + T cells with mDCs after IL10 receptor blockade. I Flow cytometry analysis of the percentage of intracellular IL4 and GATA3 in CD4 + T cells co-cultured with mDCs under different treatments. J RT-qPCR detection of mRNA expression levels of IL4 and GATA3 in CD4 + T cells co-cultured with mDCs under different treatments. K ELISA analysis of the level of IL4 in the supernatant of co-cultures of mDCs and CD4 + T cells under different treatments. L Schematic diagram of co-culture of CD4 + T cells and mDCs with IL10 antibody. M Flow cytometry analysis of the percentage of intracellular IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 antibody under different treatments; (N) RT-qPCR detection of mRNA expression levels of IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 antibody under different treatments. O ELISA analysis of the level of IL4 in the supernatant of co-cultures of mDCs and CD4 + T cells with IL10 antibody. P Schematic diagram of co-culture of CD4 + T cells and mDCs with IL10. Q Flow cytometry analysis of the percentage of intracellular IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 under different treatments. R RT-qPCR detection of mRNA expression levels of IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 under different treatments. S ELISA analysis of the level of IL4 in the supernatant of co-cultures of mDCs and CD4 + T cells with IL10; *indicates a significant difference between two groups with P < 0.05, all experiments were repeated three times

Article Snippet: Slides were incubated overnight with primary antibodies against ICAM1 (Thermofisher, MA5-43106) at a dilution of 1:60.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Control, Co-Culture Assay, Flow Cytometry

Journal: Journal of Nanobiotechnology

Article Title: New insights into allergic rhinitis treatment: MSC nanovesicles targeting dendritic cells

doi: 10.1186/s12951-024-02748-2

Figure Lengend Snippet: In vivo validation of the regulatory mechanism of P-D2-EVs. A Number of nasal itching and sneezing events within 2 h after the final intranasal OVA administration in each group of mice (n = 6). B Total cell count in NALF of each group of mice (n = 6). C OVA-specific serum IgE levels in each group of mice (n = 6). D Representative PAS staining images (scale bar = 100 μm) and quantification of PAS-positive goblet cells in nasal tissue of each group of mice (n = 6). E Representative H&E staining images (scale bar = 100 μm or 25 μm) of nasal tissue, as well as quantification of eosinophils and nasal mucosal thickness, with the red arrow indicating nasal mucosa (n = 6). F Representative images (scale bar = 25 μm) and quantification of neutrophil infiltration in nasal mucosa of each group of mice, with the black arrow indicating positive cells (n = 6). G Protein expression and quantification of Fut1, p-P38, and P3J8 in nasal tissue of each group of mice as detected by Western blotting (n = 6). H α-(1,2)-Fucose glycosylation status and quantification of ICAM1 protein in nasal tissue of each group of mice as detected by Western blotting following UEA-1 enrichment (n = 6). I Binding and quantification of ICAM1 and UEA1 in nasal tissue of each group of mice as detected by co-immunoprecipitation (n = 6). J Expression and quantification of IL10, IL4, IL13, and GATA3 in nasal tissue of each group of mice as detected by RT-qPCR (n = 6). K Levels of IL10, IL4, and IL13 in serum of each group of mice as detected by ELISA (n = 6). L Protein expression and quantification of Fut1, p-P38, and P3J8 in nasal tissue of each group of mice as detected by Western blotting (n = 6). M α-(1,2)-Fucose glycosylation status and quantification of ICAM1 protein in nasal tissue of each group of mice as detected by Western blotting following UEA-1 enrichment (n = 6). N Binding and quantification of ICAM1 and UEA1 in nasal tissue of each group of mice as detected by co-immunoprecipitation (n = 6). O Expression and quantification of IL10, IL4, IL13, and GATA3 in nasal tissue of each group of mice as detected by RT-qPCR (n = 6). P Levels of IL10, IL4, and IL13 in serum of each group of mice as detected by ELISA (n = 6); NALF nasal lavage fluid; *indicates significant difference between two groups with P < 0.05

Article Snippet: Slides were incubated overnight with primary antibodies against ICAM1 (Thermofisher, MA5-43106) at a dilution of 1:60.

Techniques: In Vivo, Cell Counting, Staining, Expressing, Western Blot, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a A schematic showing the single-cell RNA sequencing of the sorted cells from L2T- or L2G-labeled TNBC PDX (mice 1 and 2) or MDA-MB-231 tumor (mouse 3)-bearing mice, both primary breast tumors and lung metastases (early micrometastases). b Heatmap denoting expression magnitude estimates in log scale (red color) for ICAM1 and co-expressed stemness signature genes in primary tumor cells and lung metastases ( N = 3 mice, 2 with TN PDXs and 1 with MDA-MB-231 tumor). Genes are sorted according to their correlation with ICAM1 across all cells, as denoted by the gray bars on the right (top highest to bottom lowest). The bottom list of CD44, GAPDH, ACTB, eGFP, and tdTomato serve as control genes without significant changes between primary tumor cells and lung metastases. c Representative ICAM1 expression in L2T + or L2G + primary tumor cells and lung metastases determined by flow cytometry from different breast cancer PDX models (TN1, TN2, and TN3). Flow profile gates are shown in Supplementary Fig. . d Quantitative data of the differential ICAM1 expression in PDX primary tumors versus lung metastasis from c . n = 3 biological replicates (mice) for each model. One-sided t test * P = 0.04; ** P = 0.01; * P = 0.02. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). e Representative IHC staining images of ICAM1 expression (brown color) in primary tumors and lung metastases from PDX TN1 and TN2 models validating c and d . f Distribution of ICAM1 expression across PAM50 subtypes in the TCGA BRCA cohort ( N = 1037). Basal-like and HER2-enriched subtypes are the top two exhibiting significantly higher ICAM1 expression as compared to normal breast tissue (percentage of cases above the blue line value are shown for each subtype). Statistical significance was assessed using a two-sided Student’s t test. * P < 0.05, ** P < 0.01, and **** P ≤ 0.0001. g Schematic and flow histogram analyses of the orthotopically implanted TN1-PDX tumors with or without ICAM1 overexpression (OE) at the 4th mammary fat pads. h . PDX TN1 tumor weights 2 months after orthotopic injections of TN1 cells with ICAM1 OE and control vector (Con) (2.5e5 cells into one mammary fat pad/mouse). n = 3 mice per cell group. Two-sided t test * P = 0.04. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). i , j Representative lung images and normalized BLI signals (total flux) of spontaneous lung metastases in orthotopically implanted ICAM1 OE and control TN1 tumor-bearing mice as in g , h . n = 3 mice per group. Two-sided t test ** P = 0.003. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). k Schematic and flow histogram analyses of tail vein injected MDA-MB-231 tumor cells transfectd with siRNA control (siCon) and siICAM1. l , m . Representative images and quantitative data of BLI signal of mice injected with siCon and siICAM1-transfected MDA-MB-231 cells via tail vein ( n = 4 mice per cell group. Two-sided t test ** P = 0.01 for time point comparisons. N = 6 independent experiments). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum).

Article Snippet: Tissue sections were blocked with TBS/10% NGS, then incubated with ICAM1 primary antibody overnight (Sigma Aldrich, HPA004877), followed by Dako envision plus kit and DAB staining.

Techniques: RNA Sequencing Assay, Labeling, Expressing, Flow Cytometry, Whisker Assay, Immunohistochemistry, Over Expression, Plasmid Preparation, Injection, Transfection

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Representative IHC staining images of ICAM1 − single CTC and ICAM1 + CTC cluster (5-cell) (brown) in the lung vasculature of the PDX TN1-bearing mouse. Minimal 3 sets of independent images have shown similar patterns. b Representative images of CellSearch-analyzed CTCs in breast cancer (BC) patients: CD45 − , cytokeratin (CK) + (green), DAPI + (purple), ICAM1 − or ICAM1 + (gray) (two single cells and a three-cell cluster). c Bar graph of the proportion of flow analyzed CD45 − ICAM1 + single (blue) and clustered (orange) CTCs in each of 51 stage III–IV BC patients ( N = 51 patients. Two-sided t test **** P = 0.00007). d , e Representative images ( d ) and quantitative cluster counts ( e ) of ICAM1 + and ICAM1 − cells sorted from PDX TN1 OE models showing different cluster formation efficiencies ex vivo ( n = 10 biological replicates. Data are presented as mean values ± SEM. Error bars represent SE values. Two-sided t test * P < 0.02. N = 2 independent experiments). f , g Representative images ( f ) and quantified cluster sizes ( g ) of MDA-MB-231 cells transfected with siRNA control (Con) and siICAM1, and resuspended in poly-HEMA treated plates for cluster formation ( n = 10 biological replicates. Data are presented as mean values ± SD. Error bars represent SD values. Two-sided t test *** P = 0.0003. N = 4 independent experiments). h , i Diagram of solid phase self-interaction assay ( h ) and quantified binding ( i ) of biotin-conjugated ICAM1 and BSA at 1 μg to the solid phase coated with ICAM1 (1 µg), measured as OD 450 units (two-sided t test **** P = 0.0000003. N = 2 independent experiments). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). j Co-IP detection of ICAM1-Flag and ICAM-Myc intercellular homodimers. Upper panel, diagram of two HEK-293T cells transfected with C-terminal Flag-tagged and Myc-tagged ICAM1, respectively. Lower panel, immunoblots for Flag, Myc, and ICAM1, respectively, following co-IP with anti-Flag antibody. N = 3 independent experiments. k ICAM1 extracellular homodimer structure model with interacting domains between II-IV, III-III, and IV-II of two molecules extended from opposite directions. l Co-IP detection of ICAM1-Flag with ICAM-Myc variants transfected in two sets of HEK293T cells separately. Upper panel, diagram of two HEK-293T cells transfected with ICAM1-Flag (WT) and ICAM1-Myc variants (1 of 5 different variants). Lower panel, immunoblots for Flag and Myc, following co-IP with anti-Flag showing lost interactions with mutant ICAM1. Colored asterisks indicate the various WT and variant constructs ( N = 2 independent experiments).

Article Snippet: Tissue sections were blocked with TBS/10% NGS, then incubated with ICAM1 primary antibody overnight (Sigma Aldrich, HPA004877), followed by Dako envision plus kit and DAB staining.

Techniques: Immunohistochemistry, Ex Vivo, Transfection, Binding Assay, Whisker Assay, Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Variant Assay, Construct

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Down-regulated pathways upon ICAM1 knockdown in MDA-MB-231 cells, analyzed by RNA sequencing (top) and mass spectrometry analysis (bottom). b GSEA of the gene sets for histone deacetylase targets, H3K27ME3, and EZH2 targets enriched among the up-regulated genes in MDA-MB-231 ICAM1 knockdown cells in comparison to siRNA control, identified by RNA sequencing. c Immunoblots of ICAM1 and CDK6 in MDA-MB-231 cells transfected with control siRNAs (siCon) and siICAM1 for gene knockdown. N = 3 independent experiments. d , e Representative images ( d ) and mammosphere quantitation ( e ) of MDA-MB-231 cells transfected with siCon, siICAM1, and siCDK6 ( n = 3 biological replicates, two-sided t test ** P = 0.006; *** P = 0.0008. N = 3 independent experiments. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). f – h Representative images ( f ) and quantitative data ( g , h ) of BLI signals of mice injectd with siCon, siICAM1, and siCDK6-transfected MDA-MB-231 cells via tail vein ( n = 3 mice per cell group. g Data are presented as mean values ± SD, two-sided t test * P < 0.05; ** P ≤ 0.01. N = 4 independent experiments. h The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). i , j Representative images ( i ) and counts ( j ) of proliferative MDA-MB-231 (50k cells/well) transfected with siCon, siICAM1, and siCDK6. Cell numbers were measured via hemocytometer counting 48 h after seeding ( n = 4 biological replicates. two-sided t test * P = 0.04; ** P = 0.01. N = 3 independent experiments. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). k Pearson’s pairwise correlation plot of ICAM1 versus CDK6 expression in breast cancer patients ( n = 4712), by BC Gene-Expression Miner v4.4. ICAM1 vs. CDK6 R = 0.40. **** P < 0.0001.

Article Snippet: Tissue sections were blocked with TBS/10% NGS, then incubated with ICAM1 primary antibody overnight (Sigma Aldrich, HPA004877), followed by Dako envision plus kit and DAB staining.

Techniques: RNA Sequencing Assay, Mass Spectrometry, Histone Deacetylase Assay, Western Blot, Transfection, Quantitation Assay, Whisker Assay, Expressing

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Diagram of TEM assay with HUVEC cell-coated transwell inserts and tumor cells seeded on the top. Tumor cells transmigrated to the bottom were measured 24 h after seeding. (MDA-MB-231 tumor cells; green, HUVEC; purple, and ICAM1 protein; blue). b Representative images (top) and quantitative analyses (bottom) of TNBC cells transmigrated to the bottom chamber, including four conditions (from left to right): (1) siRNA control tumor cells and HUVECs; (2) ICAM1 knockdown in endothelial cells (EC); (3) ICAM1 knockdown in tumor cells; (4) ICAM1 knockdown in both tumor cells and HUVECs ( n = 3 biological replicates. Two-sided t test * P = 0.04; ** P = 0.007; *** P = 0.0006. NS not significant. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). c , d Time-lapse co-culture images at 0 and 24 h of incubation with TNBC cells (green) and endothelial cells (red) ( c ) and quantitative analyses of aggregates ( d ) following ICAM1 knockdown in both cell types ( n = 8 biological replicates. Data are presented as mean values ± SD. Error bars represent SD values. Two-sided t test **** P = 0.0000005). e , f Representative images ( e ) and quantitative analyses ( f ) of TNBC cell cluster formation in the presence of IgG control or anti-ICAM1 neutralizing antibody ( n = 10 biological replicates, Data are presented as mean values ± SEM. Error bars represent SE values. Two-sided t test ** P = 0.005). g , h Diagram and representative images of TEM assay ( g ) and quantitative analysis ( h ) of breast tumor cells transmigrated to the bottom chamber in the presence of IgG control or anti-ICAM1 neutralizing antibody ( n = 3 biological replicates. Two-sided t test ** P = 0.002). (Diagram: MDA-MB-231 tumor cells; green, HUVEC; purple, ICAM1 protein; blue, and anti-ICAM1 antibody; yellow). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum).

Article Snippet: Tissue sections were blocked with TBS/10% NGS, then incubated with ICAM1 primary antibody overnight (Sigma Aldrich, HPA004877), followed by Dako envision plus kit and DAB staining.

Techniques: Whisker Assay, Co-Culture Assay, Incubation

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Differences in distant metastasis-free survival (DMFS) of patients with breast tumors exhibiting high versus low ICAM1 expression in the GSE25055 ( N = 306) cohort. Note that patients with tumors expressing higher than median ICAM1 expression (red) are associated with significantly poorer DMFS (Logrank P = 0.035). b Differences in disease-specific survival (DSS) of patients with breast tumors exhibiting high versus low ICAM1 expression alone (left panel) or in combination with a 98-gene Stemness Signature Index (right panel) in the GSE1456-GPL96 ( N = 159) cohort. Note that patients with tumors harboring higher than median ICAM1 expression (left panel, red) exhibit significantly poorer DSS (Logrank P = 0.025). Similarly, breast cancers expressing higher than median Stemness Signature Index in addition to higher than median ICAM1 expression (Dual-High, red) exhibit poorer DSS (Logrank P = 0.034). c , d Representative images ( c ) and quantitative data ( d ) of BLI signals in mice on Day 0 (D0, 0 h) and Day 1 (D1, 24 h, dissected lungs) after injection of sorted ICAM1 + and ICAM1 − TN1 PDX cells via tail vein ( n = 4 mice per cell group. Two-sided t test ** P = 0.005). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). e – g Representative BLI images of mice at 0 h and dissected lungs at 10 h after tumor cell injections via tail vein ( e ), normalized metastatic seeding to the lungs ( f ), and L2G + CTC analysis (%) in the blood ( g ). The mice were pretreated with IgG or anti-ICAM1 neutralizing antibody (80 µg/mouse) via tail vein, and 3 h later followed by a tail vein injection of MDA-MB-231 cells (1 × 10 5 cells) and the antibody (100 μg, pre-incubated with cells for 30 min). Lungs were collected 10 h after injection. ( n = 3 mice per group. Two-sided t test ** P = 0.008; ** P = 0.01. N = 2 independent experiments. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). h , i Representative BLI images of mice on day 0 post orthotopic implantations of MDA-MB-231 tumor cells, pictures of dissected breast tumors, and BLI of dissected lungs ( h ) and quantitative lung metastasis after normalization by tumor weight of each mouse ( i ). The mice were given long-term treatment with IgG or anti-ICAM1 antibody (100 µg/mouse, twice a week for 4 weeks) ( n = 3 mice per group. Two-sided t test ** P = 0.01). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). j Diagram of ICAM1 + tumor cells initiating multicellular cluster formation in the circulation, directing TEM, and mediating lung metastasis; partially through sustained expression of CDK6. Blocking the ICAM1 intercellular homophilic interactions between tumor-tumor and tumor-endothelial cells with neutralizing antibodies (gold) will inhibit CTC cluster formation and TEM, and eventually decrease or block metastasis. (ICAM1 + tumor cells; green, ICAM1 protein; blue, ICAM1 − tumor cells; beige, Endothelial cells; purple, and anti-ICAM1 antibody; yellow).

Article Snippet: Tissue sections were blocked with TBS/10% NGS, then incubated with ICAM1 primary antibody overnight (Sigma Aldrich, HPA004877), followed by Dako envision plus kit and DAB staining.

Techniques: Expressing, Injection, Whisker Assay, Incubation, Blocking Assay