Fig 1: ICAM1 mediates tumor cell clustering through homophilic interaction.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 OD450 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).
Fig 2: Single-cell RNA sequencing profiles comparing breast cancer cells from primary and metastatic tumor sites.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. 1d. 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).
Fig 3: ICAM1 mediates transendothelial migration of breast cancer cells.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).
Fig 4: Downstream targets of ICAM1 in regulating metastasis.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.
Fig 5: ICAM1 is a therapeutic target of TNBC metastasis.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 × 105 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).
Supplier Page from MilliporeSigma for Anti-ICAM1 antibody produced in rabbit