Journal: Cancer Research Communications

Article Title: Impaired Antitumor Immune Response in MYCN -amplified Neuroblastoma Is Associated with Lack of CCL2 Secretion and Poor Dendritic Cell Recruitment

doi: 10.1158/2767-9764.CRC-21-0134

Figure Lengend Snippet: Neuroblastoma cell lines secrete few chemokines but CCL2, whose ligand CCR2 is expressed by monocytes, myeloid and plasmacytoid DCs. Chemokine arrays were used to detect chemokine secretions in supernatants recovered from confluent neuroblastoma cell lines that were incubated 24 hours. A, Raw image showing the intensities of the spots corresponding to the 38 targeted chemokines. A single experiment was performed. B, CXCL8, CXCL10, and CCL2 were measured by cytometric beads arrays in cell culture supernatant from the four neuroblastoma cell lines. Bars show the mean ± SD of four independent cultures. C, Mean fluorescence intensity of CCR2 expression was measured on PBMC from 2 healthy donors, and bars show the median and range of these two measures. D, Representative experiment (out of two) showing CCR2 expression analyzed by flow cytometry on PBMC, to define which cell subsets expressed this receptor. Blue histograms represent background with control antibody, and red histogram the specific labeling with CCR2 antibody.

Article Snippet: A positive control using recombinant human CCL2 (50 ng/mL, R&D Systems) was included, and blocking antibody directed against CCL2 (10 μg/mL, R&D Systems) was used to analyze the role of this chemokine in cell migration.

Techniques: Incubation, Cell Culture, Fluorescence, Expressing, Flow Cytometry, Labeling

Journal: Cancer Research Communications

Article Title: Impaired Antitumor Immune Response in MYCN -amplified Neuroblastoma Is Associated with Lack of CCL2 Secretion and Poor Dendritic Cell Recruitment

doi: 10.1158/2767-9764.CRC-21-0134

Figure Lengend Snippet: CCL2 induces a calcium influx in DCs, and is responsible for DC recruitment toward neuroblastoma. A, Signaling induced by CCL2 in cell subpopulations in PBMC was measured by calcium mobilization using the Fura-Red probe. Cells were loaded with Fura-Red and stained to gate specific immune cells. Calcium mobilization upon CCL2 addition (65 ng/mL) was measured by an increased fluorescence ratio (510 nm/760 nm). CCL2 induces calcium mobilization in monocytes, MDC and PDC, but not in B lymphocytes, T cells, or NK cells. Data from one representative experiment out of three. B, Transwell migration (in 2 hours) of immune cells from PBMCs toward medium, medium with CCL2, or culture supernatant from the cell lines SH and AS. CCL2 neutralizing antibody or isotype control was added to measure CCL2-driven migration. Values represent the percentage of initial cells migrating in 2 hours. Bars represent mean ± SD of two experiments, performed with 3 and 5 different healthy donors’ cells ( N = 8 healthy donors).

Article Snippet: A positive control using recombinant human CCL2 (50 ng/mL, R&D Systems) was included, and blocking antibody directed against CCL2 (10 μg/mL, R&D Systems) was used to analyze the role of this chemokine in cell migration.

Techniques: Staining, Fluorescence, Migration

Journal: Cancer Research Communications

Article Title: Impaired Antitumor Immune Response in MYCN -amplified Neuroblastoma Is Associated with Lack of CCL2 Secretion and Poor Dendritic Cell Recruitment

doi: 10.1158/2767-9764.CRC-21-0134

Figure Lengend Snippet: CCL2 level in neuroblastoma tumors correlates with DC infiltrate, role of CCL19, CCL22, and CD1c + DC in T-cell recruitment. A, Normalized expression of CCL2 and MYCN genes in the GSE62564 dataset. Tumors are separated according to the genetic amplification of MYCN (i.e., low or high). Statistical analysis: Student t test. B, Correlation plots between CCL2 expression and genes coding for specific immune population markers (specified above plots). High correlations (see Materials and Methods) in the dataset are marked in bold (Spearman correlation coefficient r and P value). C, Penalized logistic regression coefficients of 34 chemokines for regression of CD3E in the dataset GSE62564 (mean ± SD of 100 × 10-fold cross-validations). D, ROC curve and AUC of the full model defined in C , tested on dataset GSE3960. E, ROC and AUC of a logistic regression model of CD3E versus CCL19 and CCL22 trained on GSE62564 and tested on GSE3960 (blue) or GSE85047 (red). F, CCL19 and CCL22 were measured by ELISA in supernatants from cocultures of the four neuroblastoma cell lines and purified DCs activated or not by R848. Bars show the mean ± SD of three independent cultures. G, Transwell migration (18 hours) of immune cells from PBMCs toward medium, or supernatant from cocultures of the four neuroblastoma cell lines with or without DCs activated or not with R848. Values represent the percentage of initial CD3 + T cells migrating in 18 hours. Bars represent mean ± SD of three experiments, performed with different healthy donors’ DCs. H, Correlation plots between CCL19 and CCL22 expression and genes coding for specific immune population markers. High correlation in the dataset is marked in bold (Spearman correlation coefficient r and P value). MYCN status is indicated for each tumor (gray: MYCN -low, black: MYCN -high).

Article Snippet: A positive control using recombinant human CCL2 (50 ng/mL, R&D Systems) was included, and blocking antibody directed against CCL2 (10 μg/mL, R&D Systems) was used to analyze the role of this chemokine in cell migration.

Techniques: Expressing, Amplification, Enzyme-linked Immunosorbent Assay, Purification, Migration

Journal: Cancer Research Communications

Article Title: Impaired Antitumor Immune Response in MYCN -amplified Neuroblastoma Is Associated with Lack of CCL2 Secretion and Poor Dendritic Cell Recruitment

doi: 10.1158/2767-9764.CRC-21-0134

Figure Lengend Snippet: Proposed model of immune cell recruitment in neuroblastoma . MYCN -nonamplified neuroblastoma (left side) secrete CCL2, a chemokine that will initiate recruitment of APCs: monocytes and DCs (CD141 + and CD1c + MDC, and PDC). The TME in these tumors favors DC activation, and among them, CD1c + MDC will secrete CCL19 and CCL22, thereby allowing T-cell recruitment. Immune infiltration is further amplified with CCL5 secretion by T cells, and CCL21 produced by stromal cells. In MYCN -amplified neuroblastoma (right side), in the absence of CCL2 production, the tumor remains “cold.” Created with BioRender.com.

Article Snippet: A positive control using recombinant human CCL2 (50 ng/mL, R&D Systems) was included, and blocking antibody directed against CCL2 (10 μg/mL, R&D Systems) was used to analyze the role of this chemokine in cell migration.

Techniques: Activation Assay, Amplification, Produced

Journal: International journal of molecular medicine

Article Title: Role of angiotensin II type 1 receptor in cerebral aneurysm formation in rats.

doi: 10.3892/ijmm_00000239

Figure Lengend Snippet: Figure 5. Immunostaining of NF-κB, MCP-1, MMP-2 and MMP-9 in cerebral aneurysmal walls and zymography of MMP-2 and MMP-9 activities. Immunostaining of DNA binding form of NF-κB p65 subunit (C and D), MCP-1 (E and F), MMP-2 (G and H) and MMP-9 (I and J) in cerebral aneurysmal walls of rats with vehicle (C, E, G, I) or valsartan treatment (D, F, H, J). (A and B) Elastica van Gieson staining (EvG) of serial section as C (A) or D (B). Bar: 30 μm. (K) Representative image of gelatine zymography from five independent experiments.

Article Snippet: Primary antibodies used in this study are listed below: rabbit polyclonal anti-monocyte chemoattractant protein (MCP-1) antibody (Santa Cruz, Santa Cruz, CA), mouse monoclonal anti-smooth muscle ·-actin antibody (Lab Vision, Fermont, CA), goat polyclonal anti-CD68 antibody (Santa Cruz), mouse monoclonal anti-NF-κB p65 subunit antibody which recognizes only DNA-binding form (Chemicon, Temecula, CA), rabbit polyclonal anti-matrix metalloproteinase (MMP)-2 antibody (Santa Cruz), goat polyclonal anti-MMP-9 antibody (Santa Cruz), rabbit polyclonal anti-AT1R antibody (Santa Cruz), and rabbit polyclonal anti-AT2R antibody (Santa Cruz).

Techniques: Immunostaining, Zymography, Binding Assay, Staining

Journal: International journal of molecular medicine

Article Title: Role of angiotensin II type 1 receptor in cerebral aneurysm formation in rats.

doi: 10.3892/ijmm_00000239

Figure Lengend Snippet: Figure 4. Effect of valsartan on MCP-1, MMP-2 and MMP-9 mRNA expression. (A-C) Result of quantitative real-time PCR analysis of MCP-1 (A), MMP-2 (B) and MMP-9 (C) with vehicle or valsartan treatment (n=6).

Article Snippet: Primary antibodies used in this study are listed below: rabbit polyclonal anti-monocyte chemoattractant protein (MCP-1) antibody (Santa Cruz, Santa Cruz, CA), mouse monoclonal anti-smooth muscle ·-actin antibody (Lab Vision, Fermont, CA), goat polyclonal anti-CD68 antibody (Santa Cruz), mouse monoclonal anti-NF-κB p65 subunit antibody which recognizes only DNA-binding form (Chemicon, Temecula, CA), rabbit polyclonal anti-matrix metalloproteinase (MMP)-2 antibody (Santa Cruz), goat polyclonal anti-MMP-9 antibody (Santa Cruz), rabbit polyclonal anti-AT1R antibody (Santa Cruz), and rabbit polyclonal anti-AT2R antibody (Santa Cruz).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Biology

Article Title: Unveiling Lovastatin’s Anti-Inflammatory Potential in Mouse’s Brain during Acute Trypanosoma cruzi Infection

doi: 10.3390/biology13050301

Figure Lengend Snippet: Antibodies used for Western blot analysis ( Figures S1–S4 in Supplementary Materials ).

Article Snippet: Primary antibody: Anti-MCP-1 , Abcam , ab702 , 1:1000 , 20 μg.

Techniques: Western Blot, Protein Concentration

Journal: Biology

Article Title: Unveiling Lovastatin’s Anti-Inflammatory Potential in Mouse’s Brain during Acute Trypanosoma cruzi Infection

doi: 10.3390/biology13050301

Figure Lengend Snippet: Analysis of inflammatory mediators by Western blot in mice’s brain at 15 dpi. Infection led to an increase in ICAM-1 levels in YNT group; lovastatin treatment prevented this increase ( A ). Neither infection nor lovastatin treatment altered VCAM-1 levels ( B ) and MCP-1 levels ( C ). NI, non-infected; Y, infected; NT, non-treated; LOV, treated with lovastatin (20 mg/kg/day); dpi, days post infection. Asterisk compared to NINT; Sharp compared to YNT; # p < 0.05, ** p < 0.01. n = 3/4 animals per experiment in 2 independent experiments.

Article Snippet: Primary antibody: Anti-MCP-1 , Abcam , ab702 , 1:1000 , 20 μg.

Techniques: Western Blot, Infection

Journal: International Journal of Molecular Sciences

Article Title: Effects of Periostracum Cicadae on Cytokines and Apoptosis Regulatory Proteins in an IgA Nephropathy Rat Model

doi: 10.3390/ijms19061599

Figure Lengend Snippet: TLR4, MCP-1, IgA, and caspase-3 expression in rat kidney tissues. ( a ) TLR4, IgA, MCP-1, and caspase-3 expression levels were analyzed by Western blotting. ( b ) Quantitative analysis of TLR4, IgA, MCP-1, and caspase-3 protein levels. A: Healthy, normal controls; B: IgAN model group; C: prednisone acetate group; D: tripterygium glycoside tablet group; E–G: treatment group that received 0.5, 1, and 2 g/kg periostracum cicadae. Data represent the mean ± standard deviation (** p < 0.01 vs model group).

Article Snippet: Rabbit anti-TLR4 polyclonal antibody (Boster Biological Technology, Pleasanton, CA, USA), rabbit anti-IgA polyclonal antibody (Boster Biological Technology), rabbit anti-MCP-1 polyclonal antibody (Boster Biological Technology), and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (Boster Biological Technology) were used in the detection of the corresponding target proteins.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: CCL2 immunohistochemical staining in TCs and ICs. Upper row, ( A ) TCs, positive (ICs, negative); ( B ) Positive ICs with >6% CCL2 positivity (TCs, negative); Lower row, ( C ) TCs and ICs, negative; All photos are at 20× magnification.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Immunohistochemical staining, Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan–Meier analysis: Association of CCL2 staining in TCs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan–Meier analysis: Association between CCL2 expression in TCs and prognosis. Positive CCL2 expression in TCs was associated with a shorter mean OS ( p = 0.004), mean DSS ( p = 0.036), and mean RFS ( p = 0.047) than negative CCL2 expression.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Univariate and multivariate Cox’s regression analysis: Association of CCL2 staining in TCs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan–Meier analysis: Association of CCL2 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association between CCL2 expression in ICs and prognosis. Positive CCL2 expression in ICs was associated with a longer mean OS (P = 0.032), mean DSS (P = 0.001) and mean RFS (P = 0.001) than negative CCL2 expression.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Univariate and multivariate Cox’s regression analysis: Association of CCL2 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression stratified by tumor stage (pT2 vs. pT3+4) in ICs and prognosis. Analysis of patients in the pT2 group revealed that positive CCL2 expression was associated with longer mean DSS ( p = 0.033) and mean RFS ( p = 0.022) than negative CCL2 expression. Comparably for patients in the pT3+4 group, CCL2 expression was associated with longer mean DSS ( p = 0.030) and mean RFS ( p = 0.034).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression stratified by lymph node stage; (N0 vs. N1+2) in ICs and prognosis. In the N0 group, CCL2-positive patients showed a longer mean OS ( p = 0.005), mean DSS and mean RFS (both p < 0.001) than CCL2-negative patients. However, in the N1+2 group, CCL2-positive patients had a shorter mean OS ( p = 0.001), mean DSS ( p = 0.031) and RFS ( p = 0.013).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression with ICs and prognosis in the patient group not treated with chemotherapy. In the chemotherapy untreated (CT–) group, CCL2 positivity was positively associated with OS ( p = 0.012), DSS ( p < 0.001) and RFS ( p < 0.001). However, there was no association between CCL2 staining and OS, DSS or RFS in the chemotherapy-treated group (CT+).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Multivariate Cox’s regression analysis: Interactions of the parameters CCL2 staining, lymph node status, and application of chemotherapy.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression in the basal subtype in ICs and prognosis. In the basal subtype, CCL2 positive staining was associated with a better mean DSS ( p = 0.032) and mean RFS ( p = 0.044) than CCL2 negative staining. However, no association between CCL2 staining and prognosis was found in the other molecular subtypes.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing, Staining, Negative Staining