Journal: Cancers

Article Title: Drug-Induced Resistance and Phenotypic Switch in Triple-Negative Breast Cancer Can Be Controlled via Resolution and Targeting of Individualized Signaling Signatures

doi: 10.3390/cancers13195009

Figure Lengend Snippet: TNBC tissues are represented by different patient-specific signaling signatures, majority of which do not include EGFR. ( A ) Fold changes in expression levels of EGFR and pEGFR in TNBC and non-TNBC tumors are shown. Y1068 and Y1173 are EGFR phosphorylation sites; ( B ) Examples for patient-specific sets of active unbalanced processes are shown. Each sample harbors a set of 1–3 active unbalanced processes (PaSSS), represented schematically by a barcode. In each barcode active unbalanced processes are represented by black or gray squares, inactive white. Negative/positive amplitude denotes how the patients are correlated with respect to a particular process. Suggested PaSSS-based therapies appear below each barcode; ( C ) Heterogeneity index of 3 subgroups, represented by a ratio between the number of distinct PaSSSs and the number of samples in each subset, is shown for the TNBC subset of tissues, the entire set (3467 samples from 11 cancer types) and the subset of non-TNBC samples. (Abbreviations: TNBC—Triple Negative Breast Cancer, PaSSS—Patient-specific signaling signature, EGFR—Epidermal Growth Factor Receptor, VEGFR2—Vascular Endothelial Growth Factor Receptor 2, Her2—Human Epidermal growth factor Receptor 2, Src—Proto-oncogene tyrosine-protein kinase Src).

Article Snippet: The following conjugated antibodies were used: anti-p-EGFR (Y1068) (R&D Systems, Minneapolis, MN, USA, cat. no. IC3570G), anti-p-ERK2 (Thr202/Tyr204) (BioLegend, San Diego, CA, USA, cat. No. 675503), anti-p-S6 (Ser235/236) (BioLegend, cat. no. 608605), and anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA, cat. no. sc-47724AF594).

Techniques: Expressing, Phospho-proteomics

Journal: International journal of molecular sciences

Article Title: Preconditioned Chorionic Villus Mesenchymal Stem/Stromal Cells (CVMSCs) Minimize the Invasive Phenotypes of Breast Cancer Cell Line MDA231 In Vitro.

doi: 10.3390/ijms24119569

Figure Lengend Snippet: Figure 6. Modulation in expression of tumor suppressor proteins and oncogenes in MDA231 cells treated with preconditioned CVMSCs: flow cytometry analysis for the expression of tumor suppressor proteins modulated in MDA231 cells after treatment with preconditioned CVMSCs showed significant increase in the expression levels for CDH1 and IFN-gamma in both IC and CM settings as compared to untreated control (A(i)). Expression levels of oncogenes such as IDO, IL6, MMP7, and TGF-β1 reduced significantly in MDA231 cells in both IC and SF setting as compared to untreated control (B(i)). Data obtained by FACS analysis from three independent experiments were quantified and are shown in bar graphs as Mean Florescence Index (MFI) pertaining to CDH1 and IFN-gamma (A(ii)), and IDO, IL6, MMP7, and TGF-β1 (B(ii)), respectively. Bars represent standard errors. * p < 0.05.

Article Snippet: Fluorescent-labeled antibodies for flow cytometry experiments, including IFN-γ (Human IFN-gamma PE-conjugated Antibody) cat# IC285P; CDH1 (Human E-Cadherin PE-conjugated Antibody) cat# FAB18381P; IDO (Human Indoleamine 2,3-dioxygenase/IDO PE-conjugated Antibody) cat# IC6030P; IL6 (Human IL-6 PE-conjugated Antibody)cat# IC206P; MMP7 (Human MMP-7 PE-conjugated Antibody) cat# IC9071P; and TGF-β1 (Human TGF-beta 1 Alexa Fluor® 488-conjugated Antibody) cat# IC10502G were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Cytometry, Control

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: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: High NRP1 expression predicts shorter time to relapse- and distant metastasis-free survival in ER-negative breast cancer patient cohorts. a Association of NRP1 expression quartiles (Q1–Q4) with overall survival in the BrCa TCGA cohort . Data were obtained from UCSC Xena . KM Plotter analysis of relapse-free survival (left panel; RFS) and distant metastasis-free survival (right panel; DMFS) in an b unselected patient cohort (RFS; n = 4929, DMFS; n = 2765; months survival displayed as median survival), c ER-positive only (RFS; n = 3768, DMFS; n = 2016; months survival displayed as median survival) and d ER-negative only (RFS; n = 1161, DMFS; n = 749; months survival displayed as upper quartile survival) tumor subcohorts [ , ]

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 is over-expressed in the claudin-low molecular subtype of breast cancer. a Heatmap showing NRP1 expression association with PAM50, claudin-low, core claudin-low (CoreCL), ER and HER2 tumor status, as well as core claudin-low signature genes. b NRP1 mRNA expression (log2 signal) in intrinsic breast cancer subtypes and claudin-low tumors (CLDN low ) in the METABRIC patient dataset ( n = 1904), obtained through cBioPortal . c NRP1 mRNA expression across intrinsic subtypes subdivided into claudin-low (CL) and non-claudin-low tumors. Correlation of claudin-low di up-gene (CLDN low UP GES) and dii down-gene (CLDN low DN GES) GSVA-derived signature scores with NRP1 expression. Claudin-low gene signature scores were obtained via GSVA. Sample subtype is represented according to color scheme used in A-C. e NRP1 mRNA expression in METABRIC core claudin-low (CoreCL), non-core claudin-low (OtherCL) and non-claudin-low tumors . Error bars represent SEM, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing, Derivative Assay

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 expression is associated with in vivo tumor progression, cancer stemness and spheroid-initiating potential. a Western blot analysis of NRP1 expression in non-targeting control (shNT) and NRP1 shRNA-silenced (shNRP1 ) SUM159 cells inoculated into mice. b Post-inoculation tumor volumes, c tumor volume at week 8 post-inoculation and d Kaplan–Meier analysis of overall survival in SUM159 shNT and NRP1 knockdown groups. e Representative images of NRP1 immunohistochemistry in shNT and shNRP1 tumors and f quantification of NRP1 IHC staining across shNT and NRP1 knockdown groups. g NRP1 expression across CL1, CL2 and CL3 claudin-low subtypes as well PAM50 classifiers in the METABRIC dataset obtained via cBioportal [ , ]. h qPCR (left and center panel; n = 3) and Western blot (right panel) analysis of ZEB1 expression in HS578T cells after 72 h NRP1 knockdown versus NT control. i qPCR analysis of ITGA6 mRNA expression in HS578T cells (leftmost panel; NRP1 expression shown in h ) and SUM159 cells (center and right panel) after 72 h NRP1 knockdown versus NT control ( n = 3). j Western blot showing ITGA6 expression in HS578T, SUM159 and MDA-MB-231 cells after 72 h NRP1 knockdown versus NT control. k Western blot showing expression of ZEB1 and NRP1 in FACS sorted CD44 + /CD24 lo and CD44 + /CD24 hi populations of SUM159 cells. l Number of spheroids (> 50 µM) formed by day 6 following seeding of single cell SUM159 and Hs578T cell cultures containing 1,200 cells in the presence of 50 µg/ml Vesencumab (red lines) or IgG control (black lines). n = 5, along with m representative images of ( mi ) SUM159 and ( mii ) Hs578T spheroid cultures at days 4 and 6 post-seeding. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001, error bars represent SEM

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing, In Vivo, Western Blot, Control, shRNA, Knockdown, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 inhibition suppresses in vivo growth of claudin-low SUM159 orthotopic xenografts. a Luciferase-derived luminescence signal from SUM159 luc primary tumors as imaged by the IVIS Spectrum In Vivo Imaging System at 7 weeks post-tumor inoculation. Endpoint (7 weeks) mean b tumor luciferase intensity, c tumor weight and d tumor volume. e Tumor growth curves in IgG control and Vesencumab groups. f Endpoint (7 weeks) tumors from IgG control and Vesencumab groups; two additional tumors from the Vesencumab-treated group were too small to collect. g Representative images of H&E staining and Ki67 and CD31 immunohistochemistry of Vesencumab and IgG control treated tumors, with quantification of hi Ki67 and hii CD31 staining across all tumors. ‘Ves’; Vesencumab. Error bars represent SEM; ** p < 0.001; **** p < 0.0001. For a – e , n = 12–14. For g – h , n = 12

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Inhibition, In Vivo, Luciferase, Derivative Assay, In Vivo Imaging, Control, Staining, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 inhibition suppresses EGFR and PDGFRα signaling in claudin-low cells. ai Receptor tyrosine kinase array showing EGFR and PDGFRα expression in SUM159 cells after 60 min treatment with 50 μg/mL IgG or Vesencumab (top panel), or 72 h after transfection with NRP1 targeting siRNA (siNRP1 or siNRP1 ) versus non-targeting (NT) control (bottom panel), with aii corresponding densitometry. Ref1 and Ref2 represent positive controls. b Western blot showing NRP1, total EGFR, phospho-EGFR (pEGFR Y1068), total PDGFRα and phospho-PDGFRα (Y1018) expression in SUM159, MDA-MB-231 and HS578T cells after 72 h NRP1 knockdown versus NT control. c Correlation analysis between NRP1 and EGFR (right panel; Pearson: 0.24, p = 2.7e−26) and PDGFRα (left panel; Pearson: 0.52, p = 3.47e−130) mRNA expression in the METABRIC dataset ( n = 1904) . Data was obtained from cBioportal. d Immunohistochemical analysis of phosphorylated (T202/Y204) p42/44 levels in endpoint (7 weeks post-tumor inoculation) Vesencumab or IgG control treated tumors, with e representative images (right panel); scale bars = 50 µm, n = 5–6. Error bars represent SEM, * p ≤ 0.05; ** p ≤ 0.01 versus control

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Inhibition, Expressing, Transfection, Control, Western Blot, Knockdown, Immunohistochemical staining