Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence imaging of immune synapses between H1299 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. H1299 lung cancer cells are pretreated with phosphate- buffered saline (PBS) or DAC prior to coculture with γδ T cells. Quantifications of immune synapses per cancer cell on eight randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). Scale bar: 100 μm. p value is calculated by the Mann-Whitney test. ( B ) A scatter plot of DAC-induced surface proteomes in H1299 (y-axis) and A549 (x-axis) human lung cancer cells following daily treatment of 100 nM DAC for 72 hours and culture in drug-free medium for 3 days (D3R3). ICAM-1 is among the top upregulated surface proteins by DAC in both cells. ( C ) Western blot analyses of ICAM-1 protein expression in mock-treated vs. DAC-treated human lung cancer cells. D3: daily treatment of 100 nM decitabine for 72 hours. D3R3: daily treatment for 72 hours, followed by a 3-day rest period in drug-free medium. β-actin: loading control. ( D ) Immunofluorescence staining of ICAM-1 and immune synapse molecules (e.g., LFA-1, LAT) at immune synapses formed between γδ T cells and DAC-treated H1299 lung cancer cells. Scale bar: 10 μm. ( E ) Representative flow cytometric dot plot showing H1299 lung cancer cells with CRISPR-knockout of ICAM1 (KO-ICAM1) subject to γδ T cell killing for 2 hours. The effector to target (E: T) ratio is 3:1. Lung cancer cells are pre-treated with mock, DAC alone, γδ T cells alone or a combination of DAC and γδ T cells. The X-axis denotes surface ICAM1 levels. Y-axis represents signal intensities of propidium iodide. ( F ) Bar graphs showing percent cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with CRISPR-knockout of ICAM-1 subject to γδ T cell killing for 2 hours. Cell death is measured by Annexin V and propidium iodide apoptosis assays (mean ± SEM, n = 3). Statistical significance is determined by one-way ANOVA test. ( G ) Representative flow cytometric dot plot showing H1299 lung cancer cells with a Tet-on expression system of ICAM1 (OV-ICAM1) subject to γδ T cell killing for 2 hours. Doxycycline (1 μg/mL) is added 24 hours prior to coculture to induce ICAM-1 protein expression. Cell death is measured by Annexin V (x-axis) and propidium iodide (y-axis) apoptosis assays. ( H ) Bar graphs showing cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with ICAM-1 over-expression subject to γδ T cell killing for 2 hours. E:T ratio is 3:1. Cell death is measured by Annexin V and propidium iodide apoptosis assays. Statistical significance is determined by one-way ANOVA test (* p < 0.05, ** p < 0.01, ***, p < 0.001). ( I ) Immunofluorescence imaging of immune synapses between H1299 KO-ICAM1 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. Scale bar: 100 μm. Quantifications of immune synapses per cancer cell on six randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). p value is calculated by the Mann-Whitney test.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Imaging, Staining, Saline, MANN-WHITNEY, Western Blot, Expressing, Control, CRISPR, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Sequencing results of the KO-ICAM1 lung cancer cells are aligned against the reference sequence of the ICAM1 genome locus. Alignment gaps are denoted as hyphens (-) to mark the lost (knockout) regions of the edited ICAM1 genome locus.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Sequencing, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence staining of F-actin (red), ICAM-1 (green), pTyr (phosphotyrosine, white) at immune synapses between γδ T cells and DAC-pretreated H1299 lung cancer cells at D3R3. Accumulation of F-actin beneath the cell membrane is noted in DAC-pretreated lung cancer cells. DAPI: 4′,6-diamidino-2-phenylindole, as a nuclear counterstain. Scale bar: 10 μm. ( B ) Representative immunofluorescence images of the interfaces between γδ T cells and H1299 lung cancer cells (parental vs. ICAM-1 knockout (KO-ICAM1)). Signals of F-actin (red) in the periphery of H1299 cancer cells are shown in two- and-a-half-dimensional (2.5D) images in the lower panels. Scale bar: 10 μm. ( C ) Dot plots of signal intensities of F-actin (left panel) and ICAM-1 (right panel) from five pTry-positive immune synapses between γδ T cells and H1299 lung cancer cells (parental or KO-ICAM1). p value is calculated by two-way ANOVA test. ( D ) Immunofluorescence images of immune synapses between γδ T cells (marked with T) and H1299 lung cancer cells (marked with C) stained for ICAM-1 (green), F-actin (Red) and phosphotyrosine (pTyr, white). Lung cancer cells (parental or KO-ICAM1) are pretreated with PBS (Mock) or 100 nM DAC and cocultured with γδ T cells at D3R3. ( E ) Dot plots of F-actin signal intensities at immune synapses between γδ T cells and H1299 cells. H1299 cells are pretreated with PBS (Mock), DAC alone or combination of DAC pretreatment (D3R3) and 1 μg/mL Cyto B (cytochalasin B, an inhibitor of actin filament polymerization) for 1.5 hours prior to coculture with γδ T cells (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (***, p < 0.001; ****, p < 0.0001). ( F ) Representative immunofluorescence images of immune synapses (pTyr staining) between γδ T and H1299 cells pretreated with PBS (Mock), DAC alone, and combination of DAC and Cyto B. Blow-up images of the square areas for each treatment are shown in the lower panels. Arrows denote immune synapses between γδ T and H1299 cells. Scale bar: 100 μm (upper) and 20 μm (lower panels). ( G ) Dot plots showing numbers of immune synapses per cancer cell on eight randomly taken high power fields for H1299 cells pretreated with PBS (Mock), DAC, and combination of DAC and Cyto B (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (*, statistical significance).

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Staining, Membrane, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Parental or ICAM-1 knockout (KO-ICAM1) H1299 cells are pretreated daily with PBS (Mock) or 100 nM DAC for 72 hours followed by 3-day drug-free culture before coculture with γδ T cells. Signal intensities of each protein (F-actin, red; ICAM-1, green; phosphotyrosine, pTyr, white) along the immune synapse area are graphed on the right. DAPI: 4’,6-diamidino-2-phenylindole, as nuclear counterstain. Scale bar: 10 μm.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Visualization of multi-omics data (i.e., mRNA-seq, Omni-ATAC-seq, and MethylationEPIC arrays) for DAPK3 , EVPLL, and TUBE1 in H1299 lung cancer cells. ( B ) Promoter methylation status and mRNA expression levels of the ICAM1 gene measured by Infinium MethylationEPIC arrays (left panels) and mRNA-seq (right panels) in human lung cancer cells treated without and with DAC 100 nM DAC for 3 days followed by a 3-day drug-free culture. ( C ) Open chromatin regions in the promoter areas of the ICAM1 gene in human lung cancer cells upon 100 nM DAC treatment analyzed by Omni-ATAC-seq. The green bar represents a CpG island. ( D ) Validation of Omni-ATAC-seq by quantitative real-time PCR on transposase-accessible chromatin at the ICAM1 promoter of human lung cancer cells subject to daily treatment of 100 nM DAC treatment for 3 days, followed by a 3-day drug-free culture. Experiments are performed in triplicates, and data are presented as mean ± SD. p value was calculated by unpaired t test (*, p < 0.05). ( E ) IPA Network analysis of mRNA expression changes in human lung cancer cells treated by DAC reveals coordinated changes of the immune-related surface molecules and the cytoskeleton-associated genes. ( F ) IPA upstream regulator analysis of mRNA expression changes in human lung cancer cells treated by DAC. T cell effector cytokines such as TNF-α and IFN-γ may enhance DAC-induced expression changes of immune-related molecules and ICAM-1 in lung cancer cells. TP53 is a potential master regulator for cancer cytoskeleton reorganization essential for DAC-potentiated γδ T cell killing.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Biomarker Discovery, Methylation, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Diagram of transcription factor binding sites at the ICAM1 promoter derived from the ENCODE ChIP-seq data ( https://www.encodeproject.org ). Visualizations of ATAC-seq peaks at the ICAM1 promoter in PC9 and CL1-5 lung cancer cell lines subject to DAC treatment are shown above. ( B ) Promoter methylation status and mRNA expression levels of putative transcription factors (i.e., RELB, NFKB2, STATS, and RUNX3) at the ICAM1 promoter in A549, H1299, PC9, and CL1-5 lung cancer cells. Dot and line plots represent methylation levels (β values) of promoter probes measured by Infinium MethylationEPIC arrays. The promoter probes with β values greater or equal to 0.5 at baseline (Mock) are shown. Bar graphs represent relative mRNA expression levels based on normalized FPKM measured by mRNA-seq.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Binding Assay, Derivative Assay, ChIP-sequencing, Methylation, Expressing

Journal: STAR Protocols

Article Title: Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility

doi: 10.1016/j.xpro.2019.100013

Figure Lengend Snippet: Mechanical Rigidity of ICAM1 Nano-Topographies Controls T Cell On-Ridge Spreading and In-Groove Invasiveness Plasticity Balance (A) 3D super-resolution reconstruction of the ICAM1-functionalized PAA nano-topographic surface (G’=16 kPa). (B) Test super-resolution imaging (3D reconstruction) of human CD4+ T cells spreading and migrating along the ICAM1-coated nano-topographic surfaces on soft (16 kPa) and rigid (50 kPa) PAA surfaces. (C) Schematic of the T cell morphometric analysis and metrics: T cell height, projected area of spreading of entire cell interface ( S entire cell IF ) and projected area of in-groove invasive T cell interface ( S invasive IF ). (D and E) Mechanoregulation of T cell height, spreading area and invasiveness as indicated by T cell spreading assay on soft (G’=16 kPa) and rigid (G’=50 kPa) ICAM1 nano-textures. T cell spreading enhances on the rigid ICAM1, accompanied with T cell flattening, i.e. decrease of the T cell height. Results indicate a mechanically controlled dynamic balance between on-ridge T cell spreading and in-groove invasiveness, as shown on the schematic panel (E). I.e. T cell in-groove invasiveness structurally competes with on-ridge spreading, indicating that on-ridge spreading is mechanically enhanced and out-balances in-groove invasiveness on the rigid (G’=50 kPa) ICAM1 nano-topography. Alternatively, soft (G’=16 kPa) ICAM-1 nano-textures are unable to promote the mechanically sensitive on-ridge T cell spreading, shifting the balance towards steric in-groove T cell invasiveness. Data on the plots on (D) are as follows: boxes - means, Q1 and Q3; whiskers - max and min, X - medians; p values - one way ANOVA test. Experimental data collected in triplicates, total n>50.

Article Snippet: ICAM1, Human Protein, Recombinant, hIgG1-Fc.His Tag, Active , Sino Biological, China , Cat#10346-H03H-50.

Techniques: Imaging

Journal: STAR Protocols

Article Title: Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility

doi: 10.1016/j.xpro.2019.100013

Figure Lengend Snippet:

Article Snippet: ICAM1, Human Protein, Recombinant, hIgG1-Fc.His Tag, Active , Sino Biological, China , Cat#10346-H03H-50.

Techniques: Recombinant, Electrophoresis, Concentration Assay, Labeling, Saline, Modification, Cell Isolation, Derivative Assay, Software

Journal: International Journal of Cardiology. Heart & Vasculature

Article Title: Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application

doi: 10.1016/j.ijcha.2015.01.004

Figure Lengend Snippet: Cell density of endothelial cells cultivated in the presence of soluble CD54. Endothelial cells cultivated in the presence of soluble CD54 (sCD54) were expanded for five splits. After the first (p1), second (p2) and fifth (p5) passages (as shown in the x-axis) the cellular densities were measured (y-axis) and compared between different growing conditions: in the presence of sCD54 alone, resulting in higher propagation rate, than cell culture treated with HUVEC-conditioned medium collected at p4 (CMP4) alone.

Article Snippet: Recombinant human ICAM-1/CD54 (murine myeloma cell line, NSO-derived; Accession #CAA30051), CD146, KDR and CD31 were purchased from R&D Systems, Inc.; CD45 was purchased from AbD Serotec, CD34 and CD14 were purchased from Becton Dickinson Pharmingen, and fibronectin was purchased from Southern Biotech.

Techniques: Cell Culture

Journal: International Journal of Cardiology. Heart & Vasculature

Article Title: Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application

doi: 10.1016/j.ijcha.2015.01.004

Figure Lengend Snippet: Capillary density. Tube formation occurred through an ordered sequence of events and was investigated with an inverted optical light microscope; (A) cells beginning to migrate and align themselves to close polygons beginning (B) to form complete tubules (C) of endothelial cells cultivated with the addition of sCD54. The histogram shows the data mean ± SD of the quantitative analysis of tube formation area; in the x-axis was reported capillary density in the presence of HUVEC-conditioned medium collected at p4 (CMP4), sCD54 and antibody direct against CD54 at the concentration of 15 ng/ml (y-axis). Scale bars: 200 μm.

Article Snippet: Recombinant human ICAM-1/CD54 (murine myeloma cell line, NSO-derived; Accession #CAA30051), CD146, KDR and CD31 were purchased from R&D Systems, Inc.; CD45 was purchased from AbD Serotec, CD34 and CD14 were purchased from Becton Dickinson Pharmingen, and fibronectin was purchased from Southern Biotech.

Techniques: Sequencing, Light Microscopy, Concentration Assay