Journal: Hepatology (Baltimore, Md.)

Article Title: Intratumoral γδ T-CellInfiltrates, Chemokine (C-C Motif) Ligand 4/Chemokine (C-C Motif) Ligand 5 Protein Expression and Survival in Patients With Hepatocellular Carcinoma

doi: 10.1002/hep.31412

Figure Lengend Snippet: Gene expression profiles of HCC tumor tissues stratified by γδ T-cell gene signature predicted low- and high-risk subgroups. (A) Hierarchical clustering of the 974 differentially expressed genes (P < 0.001) between γδ T-cell-signature–predicted subgroups. Pseudocolors indicate transcript levels above (blue), below (yellow), or equal to (black) the mean, respectively. Genes were ordered by centered correlation and complete linkage. The scale represents gene expression level from −3.0 to 3.0 in a log2 scale. (B) Result of GSEA enrichment of chemokine signal pathway based on the genes highly expressed in the γδ T high-tumor subgroup. (C) Pearson’s correlation analysis between CCL4, CCL5, and CCR5 expression level and γδ T-cell weights in HCC tumor tissues from the LCI cohorts. (D) Comparison of expression of CCL4, CCL5, and CCR5 in the subgroups defined by γδ T-cell signature in tumor tissues from the LCI cohorts. Abbreviation: KEGG, Kyoto Encyclopedia of Genes and Genomes. ***P < 0.001.

Article Snippet: The IHC score of CCL5 was calculated by multiplying the percentage of cells positively stained (0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) by the intensity of staining (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong) with a maximum of 300. ( 18 ) γδ T CELLS MIGRATION ASSAY Chemotaxis of γδ T cells was analyzed in transwell migration assays. γδ T cells were expanded and sorted from splenocytes as described. ( 19 ) Next, 5 × 10 5 γδ T cells were taken up in RPMI 1640 and placed into 5-mm pore-size transwell inserts (Corning, NY), which were placed into 24 wells of a tissue-culture plate containing 5 × 10 5 Hepa1–6 cells with 0.5 μg/mL of anti-CCL5 (R&D MAB478; R&D Systems, Minneapolis, MN) or 3 μg/mL of anti-CCL4 (R&D MAB451).

Techniques: Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Intratumoral γδ T-CellInfiltrates, Chemokine (C-C Motif) Ligand 4/Chemokine (C-C Motif) Ligand 5 Protein Expression and Survival in Patients With Hepatocellular Carcinoma

doi: 10.1002/hep.31412

Figure Lengend Snippet: Roles of tumor-cell–derived CCL4/5 on γδ T cells. (A) Spearman’s correlation analysis between CCL5 expression level and TCRγδ-positive staining cells number in the 182 FFPE HCC tissues. (B) Comparison of CCL5 expression levels between the high and low γδ T-cell-infiltrating groups. (C) Results of transwell migration assay, reflected by the proportion of chemo-attracted γδ T cells by murine hepatoma cell line Hepa1–6 with or without neutralized anti-CCL4/5. (D) Comparison of IFN-γ expression levels in transmigrated and unmigrated γδ T cells in the coculture model with Hepa1–6. (E,F) B6 mice were treated with intrahepatic trasplantation of either CCL5 shRNA-transduced or control oligo-transduced Hepa 1–6. At day 14, tumor-bearing livers were excised and used for the analysis of γδ T-cell proportion by flow cytometry (E) or analysis of expression levels of IFN-γ and perforin by real-time RT-PCR (F). Abbreviation: shCCL5, chemokine (C-C motif) ligand 5 with short hairpin RNA. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The IHC score of CCL5 was calculated by multiplying the percentage of cells positively stained (0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) by the intensity of staining (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong) with a maximum of 300. ( 18 ) γδ T CELLS MIGRATION ASSAY Chemotaxis of γδ T cells was analyzed in transwell migration assays. γδ T cells were expanded and sorted from splenocytes as described. ( 19 ) Next, 5 × 10 5 γδ T cells were taken up in RPMI 1640 and placed into 5-mm pore-size transwell inserts (Corning, NY), which were placed into 24 wells of a tissue-culture plate containing 5 × 10 5 Hepa1–6 cells with 0.5 μg/mL of anti-CCL5 (R&D MAB478; R&D Systems, Minneapolis, MN) or 3 μg/mL of anti-CCL4 (R&D MAB451).

Techniques: Derivative Assay, Expressing, Staining, Transwell Migration Assay, shRNA, Flow Cytometry, Quantitative RT-PCR

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 1. Binding and in vitro activity of murine 18V4F hybridoma antibody. (A) ELISA binding of original 18V4F hybridoma antibody to a panel of chemokines (determined in triplicate, shown as mean +/2 standard deviation). Directly coated chemokines were used here for direct comparisons, however in other experiments CCL3 showed a significantly enhanced signal when biotinylated and coated on streptavidin plates. (B) Chemotaxis inhibition by 18V4F hybridoma antibody of CCR5-transfected Ba/F3 cells to 5 ng/mL of CCL3, CCL4, and CCL5. Data are representative of at least three similar experiments. All chemotaxis data are represented as a percent of maximum migration in the absence of inhibitors and is fit using a standard four parameter dose-response model (GraphPad). doi:10.1371/journal.pone.0043332.g001

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Binding Assay, In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Chemotaxis Assay, Inhibition, Transfection, Migration

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 2. Diagram of phage display selection strategy. Individual CDR libraries were sequentially panned against CCL3, CCL4 and CCL5. In step 1, phage libraries were combined with biotinylated CCL3 and bound to streptavidin beads. Bound phage were eluted, amplified, and subjected to panning against biotinylated CCL4 and CCL5 in steps 2 and 3, respectively. This process was repeated 4–5 times with increasing stringency to yield sequences with improved affinities. doi:10.1371/journal.pone.0043332.g002

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Selection, Amplification

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 3. Chemotaxis inhibition by affinity matured 18V4F variants. Chemotaxis inhibition by humanized 18V4F Fab, d5 variant, d7 variant, d5d7, and a negative control Fab of CCR5-transfected Ba/F3 cells to 5 ng/mL of (A) CCL3, (B) CCL4, and (C) CCL5. Data are representative of at least two similar experiments. A loss in potency of humanized 18V4F Fab was observed compared with the 18V4F hybridoma shown in Figure 1b and is likely a result of both the humanization process and loss in avidity caused by switching from full IgG to Fab fragment. doi:10.1371/journal.pone.0043332.g003

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Chemotaxis Assay, Inhibition, Variant Assay, Negative Control, Transfection

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 4. Comparison of vCCI and d5d7 binding epitopes. Competitive binding ELISA examining molecules that can disrupt the d5d7-CCL3 binding interaction using d5d7 as a homologous competitor and vCCI-Fc, commercial anti-CCL3 antibody, and control IgG as heterologous competitors. Data are representative of at least two similar experiments. Competition experiments were also completed to analyze the d5d7-CCL4 and d5d7-CCL5 binding interactions and similar binding competition was observed between d5d7 and vCCI-Fc (data not shown). doi:10.1371/journal.pone.0043332.g004

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 5. Inhibition of chemotaxis induced with mixtures of chemokines by MAb d5d7. Inhibition of chemotaxis of (A) CCR5 transfectants to a pool of recombinant CCL3, CCL4, and CCL5 and (B) CCR1 transfectants to a pool of CCL3 and CCL5, by MAb d5d7 antibody, vCCI-Fc, individual commercial anti-chemokine antibodies (anti-CCL3, anti-CCL4, and anti-CCL5), and IgG controls. Chosen chemokine concentrations were those that produced 50% maximal chemotaxis when tested individually (a pool of 3 ng/mL CCL3, 10 ng/mL CCL4, and 3 ng/mL CCL5 was used in CCR5 experiments and a mixture of 20 ng/ mL CCL3 and 5 ng/mL CCL5 was used in CCR1 experiments). doi:10.1371/journal.pone.0043332.g005

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Inhibition, Chemotaxis Assay, Recombinant, Produced

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 8. SCID-hu mouse model of leukocyte migration. (A) NSG (NOD/SCID/IL2r-cnull) mice were injected i.v. with human PBMC and allowed to engraft for 10 d. MAb d5d7 was administered i.v. just before chemokines were injected s.c. in Matrigel. After 7 d the skin sites were harvested and single cell suspensions were generated. Human leukocytes were tagged with specific antibodies and analyzed by flow cytometry. (B) Inhibition by MAb d5d7 of skin leukocyte migration into chemokine-embedded Matrigel plugs in NSG mice engrafted with human PBMC. The negative control group consisted of animals treated with s.c. injection of Matrigel + PBS and i.v. administration of control IgG. All other groups had s.c. injections of Matrigel containing CCL3, CCL4, and CCL5 (400 ng each) with i.v. administration of PBS, control IgG, or MAb d5d7 antibody. Data were analyzed using a student t test. doi:10.1371/journal.pone.0043332.g008

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Migration, Injection, Generated, Flow Cytometry, Inhibition, Negative Control, Control

Journal: Frontiers in Immunology

Article Title: Importance of Salmonella Typhi-Responsive CD8+ T Cell Immunity in a Human Typhoid Fever Challenge Model

doi: 10.3389/fimmu.2017.00208

Figure Lengend Snippet: CD8+ T cell responses against Salmonella enterica serovar Typhi ( S . Typhi) at baseline predict the clinical outcome after challenge . PBMC isolated at baseline from each participant (TD n = 13, blue; NoTD n = 7, red) were stimulated for 18 h with S . Typhi-infected AEH cells (squares), S . Typhi-infected B-EBV cells (circles), or S . Typhi-infected blasts (triangles). After co-culture, cells were immunostained with a 14-color panel of mAbs and analyzed by follow cytometry as described in Section “ .” Each symbol represents the net percentage of positive cells measured for CD107a, IFN-γ, TNF-α, MIP-1β, interleukin (IL)-17A, and IL-2 in the CD8+ T EM , T effector memory CD45RA+ (T EMRA ), and T central memory (T CM ) subsets as indicated. Statistical analyses were performed using mixed effects models to account for multiple observations per person. * p < 0.05; ** p < 0.01.

Article Snippet: After co-culture with stimulator cells, PBMC were harvested, washed in 1× PBS, stained extracellularly, permeabilized, and stained intracellularly, using a 14-color panel containing Yellow Live/Dead viability kit (Invitrogen, Carlsbad, CA, USA) and mAb to CD14-BV570 (M5E2, Biolegend), CD19-BV570 (HIB19, Biolegend), CD3-BV650 (OKT3, Biolegend), CD4-PECy5 (RPA-T4, BD), CD8-PerCP-Cy5.5 (SK1, BD), CD45RA-biotin (HI100, BD), CD62L-APC-A780 (DREG-56, Ebioscience), integrin α 4 β 7 -A647 (ACT1; conjugated in house) streptavidin(SAV)-Qdot800 (Invitrogen) CD69-ECD (TP1.55.3, Beckman Coulter), IFN-γ-PE-Cy7 (B27, BD), TNF-α-A700 (MAb11, BD), IL-2-BV605 (MQ1-17H12, Biolegend), IL-17A-BV421 (BL168, Biolegend), and MIP-1β-PE (IC271P, R&D) as previously described ( ).

Techniques: Isolation, Infection, Co-Culture Assay, Cytometry

Journal: Frontiers in Immunology

Article Title: Importance of Salmonella Typhi-Responsive CD8+ T Cell Immunity in a Human Typhoid Fever Challenge Model

doi: 10.3389/fimmu.2017.00208

Figure Lengend Snippet: High levels of CD8+ T cell responses against Salmonella enterica serovar Typhi ( S . Typhi) are associated with delayed time to diagnosis . After stimulation of PBMC with S . Typhi-infected AEH cells, percentages of CD107a, IFN-γ, TNF-α, MIP-1β, interleukin (IL)-17A, and IL-2-positive cells in the CD8+ T EM and T effector memory CD45RA+ (T EMRA ) subsets were plotted against time to diagnosis for each participant who developed typhoid fever ( n = 13). Correlations (red lines) were assessed using the linear regression function and Pearson’s tests of GraphPad Prism v7.02. * p < 0.05.

Article Snippet: After co-culture with stimulator cells, PBMC were harvested, washed in 1× PBS, stained extracellularly, permeabilized, and stained intracellularly, using a 14-color panel containing Yellow Live/Dead viability kit (Invitrogen, Carlsbad, CA, USA) and mAb to CD14-BV570 (M5E2, Biolegend), CD19-BV570 (HIB19, Biolegend), CD3-BV650 (OKT3, Biolegend), CD4-PECy5 (RPA-T4, BD), CD8-PerCP-Cy5.5 (SK1, BD), CD45RA-biotin (HI100, BD), CD62L-APC-A780 (DREG-56, Ebioscience), integrin α 4 β 7 -A647 (ACT1; conjugated in house) streptavidin(SAV)-Qdot800 (Invitrogen) CD69-ECD (TP1.55.3, Beckman Coulter), IFN-γ-PE-Cy7 (B27, BD), TNF-α-A700 (MAb11, BD), IL-2-BV605 (MQ1-17H12, Biolegend), IL-17A-BV421 (BL168, Biolegend), and MIP-1β-PE (IC271P, R&D) as previously described ( ).

Techniques: Infection

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Chemokines and chemokine receptors expression in the lesions of patients with American cutaneous leishmaniasis

doi: 10.1590/0074-0276108042013008

Figure Lengend Snippet: chemokines and chemokine receptors in lesions of different clinical forms of American cutaneous leishmaniasis. A, B: density of CXCL10, CCL4, CXCR3 and CCR5 positive cells; C, D: density of CCL11, CCL8 and CCR3 positive cells; E: density of CXCL8; F: CCR7 positive cells. Data are represented as medians and interquartile range patients with localised cutaneous leishmaniasis (LCL) (n = 20), intermediate cutaneous leishmaniasis (ICL) (n= 5) and diffuse cutaneous leishmaniasis (DCL) (n = 10). Asterisk means p < 0.05 comparing LCL with DCL, LCL with ICL and LCL with ICL by the Mann-Witney U test.

Article Snippet: For chemokine characterisation of frozen sections, affinity-purified goat polyclonal antibodies directed to the following human chemokines were used: CXCL10 (IP-10) (sc-1406, Santa Cruz Biotechnology Inc, California, USA) (2 μg/mL), CCL8 (MCP2) (sc-1307, Santa Cruz Biotechnology Inc, California, USA) (2 μg/mL), CCL4 (MIP1β) (AF-271-NA, R&D Systems Inc, USA) (5 μg/mL) and CXCL8 (IL-8) (AF-208-NA, R&D Systems, Inc, USA) (2.5 μg/mL).

Techniques:

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 3. Macrophage-assisted ECM degradation and invasion by cancer cells is mediated by MIP-1b. (A and B) Representative images showing the effect of anti-human MIP-1b blockade by MIP-1b-neutralizing antibody (MIP-1b NA) or addition of MIP-1b-purified cytokine (MIP-1b) with respect to matrix protrusive activity, and invasion in MDA-MB-231 and MCF-7. IgG serve as isotype antibody control for MIP-1b-neutralizing antibody (MIP-1b NA). Cancer cells (MDA-MB-231 and MCF-7) treated with MIP- 1b NA showed decreased activity in matrix protrusive activity and diminished invasion compared to cells that were not treated with MIP-1b NA. Cancer cells treated with MIP-1b-purified cytokine (MIP-1b) showed an increase in matrix protrusive activity and invasion compare to cells that did not treated with MIP-1b. Bars represent mean invadopodia count/cell from 10 fields per experiment and mean invasive cell count §SE (p < 0.05.). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer Cells; CCM: Respective cancer cells co-cultured withMacrophages; CCM(IgG): Respective cancer cells co-cultured with macro- phages treated with isotype antibody control IgG; CCMCMIP-1b NA: Respective cancer cells co-cultured with macrophages treated with MIP-1b-neutralizing antibody; MIP-1b: Respective cancer cells treated with MIP-1b-purified cytokine.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Activity Assay, Control, Cell Counting, Cell Culture

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 4. Macrophage-assisted ECM degradation and invasion by cancer cells is mediated by MIP-1b via its receptor. (A and B) Silencing MIP-1b cognate recep- tors viz. CCR4 and CCR5 abrogated macrophage induced in vitro ECM degradation, and invasion by cancer cell. Cancer cells (MDA-MB-231 and MCF-7) silenced with CCR4 and CCR5 showed decreased ECM degardation and invasive activity compared to cells having scrambled in both the conditions viz. cancer cells co- cultured with macrophages [CCM] and cultured alone [C]. Bars represent mean invadopodia count/cell from 10 fields per experiment and mean invasive cell count §SE (p < 0.05.). Silencing was confirmed by protein gel blot. All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; CCM: Respective cancer cells co-cultured withmacrophages.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: In Vitro, Activity Assay, Cell Culture, Cell Counting, Western Blot

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 5. Blockade of MIP-1b function minimized ex ovo dissemination of co-cultured cancer cells from chick chorioallantoic memebrane to chik brain. (A and B) Repre- sentatitive results of ex ovo chick chorioallantoic membrane assay for spontaneous metastasis. MIP-1b-neutralizing antibody (MIP-1b NA) mediated in vivo functional blockade of MIP-1b-impeded spontaneous metastasis of co-cultured (with macrophages) breast cancer cells [C C M C MIP-1b NA] compare to cells that were not treated with MIP-1b NA [C C M C IgG and C C M] from chicken chorioallantoic membrane to chicken brain. Bars represent number of fluorescent foci in each group. All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; C C M: Respective cancer cells co-cultured with macro- phages; C C M(IgG): Respective cancer cells co-cultured with macrophages treated with isotype antibody control IgG; C C M C MIP-1b NA: Respective cancer cells co-cul- tured with macrophages treated with MIP-1b-neutralizing antibody.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Cell Culture, Chick Chorioallantoic Membrane Assay, In Vivo, Functional Assay, Membrane, Control

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 6. TAMs-assisted cancer cell invasion via MIP-1b is dependent on upregulation of MYO3A gene with in cancer cells. (A, B and C) Quantitative RT-PCR- based validation of selected genes (through c-DNA-based gene-expression analysis) in mono-cultured [C] and co-cultured (with macrophages) cancer cells [CCM]. Bars represent relative fold change in expression levels §SE (p < 0.05). (D, E and F) MYO3A gene exhibited a characteristic MIP-1b-responsive mRNA exprerssion profile. MIP-1b-neutralizing antibody (MIP-1b NA) treated cells showed decrease in MYO3A expression compared to cells that were co-cultured with macrophages [CCM] and/or IgG antibody control [C C M C IgG]. MIP-1b-purified cytokine (MIP-1b) enhanced the expression of MYO3A gene in monocultured cancer cells compared to cells that were not treated with MIP-1b-purified cytokine (C). Bars represent Quantitative RT-PCR relative fold change expression §SE (p <0.05.). All the experiments were done in triplicates and repeated at least thrice. (G and H) Blockade of MIP-1b with MIP-1b-neutralizing antibody (MIP-1b NA, Fig. 6G) and its cognate receptor CCR4 and CCR5 silencing (CCR4-siRNA and CCR5-siRNA, Fig. 6H)) downregulated expression levels of MYO3A in breast cancer cells (MDA-MB-231, MDA-MB-468 and MCF-7). MIP-1b-purified cytokine (MIP-1b) enhanced the expression of MYO3A gene in monocultured cancer cells compared to cells that were not treated with MIP-1b-purified cytokine (C) Fig. 6G. All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; C C M: Respective cancer cells co-cultured with macrophages; CCM(IgG): Respective cancer cells co-cultured with macrophages treated with isotype antibody control IgG; C C M C MIP-1b NA: Respective cancer cells co-cultured with macrophages treated with MIP-1b-neu- tralizing antibody; MIP-1b: Respective cancer cells treated with MIP-1b-purified cytokine.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Cell Culture, Expressing, Control

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 7. MYO3A serves as a key cancer cell intrinsic effector for macrophage-assisted cancer cell invasion via MIP-1b. TAMs-assisted cancer cell invasion via MIP-1b is dependent on upregulation of MYO3A gene within cancer cells. (A and B) Macrophage-induced matrix protrusion and invasion by cancer cells was abrogated upon siRNA-mediated silencing of MYO3A gene compare to scrambled in both C (mon-oculture cancer cells) and C C M (cancer cells co-cultured with macrophages). Bars repre- sent mean invadopodia count/cell from 10 fields per experiment and mean invasive cell count §SE (p < 0.05.). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; CCM: Respective cancer cells co-cultured withmacrophages.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Cell Culture, Cell Counting

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 8. Invasive breast adenocarcinoma MDA-MB-231 silenced with MYO3A exhibited diminished focal degradation of pericellular matrix, diminished invadopodia for- mation, compared to Scrambled control in presence of MIP-1b-Purified cytokine. Representative images from the in vitro matrix degradation assay. Cells (MDA-MB-231) Scr and silenced with MYO3A siRNA were seeded on Alexa Fluor 633 labeled gelatin (Red) in absence or presence of MIP-1b-purified cytokine (MIP-1b) for 24 h, followed by fixation, staining with Alexa fluor 488 phalloidin (Green) and mounting in aqueous media containing DAPI (Blue). Compared to Scr control MYO3A-directed siRNA, MDA-MB-231 cancer cells did not show any effect of MIP-1b-purified cytokine on focal degradation of pericellular matrix. Bars represent mean invadopodia count/cell from 10 fields per experiment §SE (p < 0.05.). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—MIP-1b: Respective cancer cells treated with MIP-1b-purified cytokine.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Control, In Vitro, Degradation Assay, Labeling, Staining

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 9. Diminished expression of MMP-9 and MYO3A upon neutralizing antibody-mediated blockade of MIP-1b function was followed by reduced cellular burden in lungs and diminished presence of metastatic foci in liver of syngenic 4T1/BALB/c mouse model of breast cancer. (A and B) Anti-mouse MIP-1b goat IgG polyclonal anti- body-treated 4T1 tumors expressed much lower invasive potential as revealed by significantly downregulated mRNA expression levels of MYO3A. The mRNA expression levels of MMP-9 gene were significantly downregulated. Bars represent Quantitative RT-PCR relative fold change expression §SE (p <0.05). (C): On day 26th post graft- ing, compared to controls (PBS or isotype control antibody (IgG)), the intratumoral administration of MIP-1b-neutralizing antibody (MIP-1b NA) resulted in reduced cellu- lar burden in lungs and perivascular regions of liver from 4T1/BALB/c mouse models. Lung and liver sections obtained from healthy uninoculated mice served as mock control. Bars represent no. of metastatic lesions §SE (p < 0.05.). All the experiments were done in triplicates. All the experiments were done in triplicates. Abbrevia- tions—4T1 tumor (PBS): 4T1-induced tumor treated with PBS; 4T1 tumor (IgG): 4T1-induced tumor treated with isotype control antibody; 4T1 Tumor (MIP-1b NA): 4T1- induced tumor treated with MIP-1b-neutralizing antibody.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 11. Association between fold change (tumor tissue vs. matched normal) in mRNA expression level of MIP-1b, MYO3A, and MMP-9 in human breast cancer speci- men. (A and B) Distribution of breast cancer patients (n D `50) with respect to fold change in expression of selected markers and correlation between fold expressions of marker genes of breast cancer patients (n D 50). (C) Association of MYO3A and MIP-1b expression with overall survival. RT-PCR analysis was done in triplicates and repeated at least thrice.

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: OncoImmunology

Article Title: Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation ofMYO3Agene in breast cancer cells

doi: 10.1080/2162402x.2016.1196299

Figure Lengend Snippet: Figure 12. Schematic representation of the study. Macrophages-derived cytokine MIP-1b enhances the expression of MYO3A in cancer cells that markedly increase ECM degradation and invasive potential of breast cancer cells. These cascade of events culminate into distant metastasis (Fig. 12).

Article Snippet: Recombinant human MIP-1b (271-BME/CF) and anti-human MIP-1b goat IgG polyclonal antibody (AB-271-NA) and antimouse MIP-1b goat IgG polyclonal antibody (AB-451-NA) were from R&D systems (USA).

Techniques: Derivative Assay, Expressing