ccl2 neutralizing antibody  (R&D Systems Hematology)

Journal: Oncoimmunology

Article Title: Glutamate promotes CCL2 expression to recruit tumor-associated macrophages by restraining EZH2-mediated histone methylation in hepatocellular carcinoma

doi: 10.1080/2162402X.2025.2497172

Figure Lengend Snippet: Glutamate increases CCL2 expression on hepatoma cells.

Article Snippet: For the CCL2-CCR2 studies, mice were treated with CCL2 neutralizing antibody (2 mg/kg, Bio X cell, cat#BE0185) or CCL2 mAb in combination with MK801 intraperitoneally twice a week for 2 weeks.

Techniques: Expressing

Journal: Oncoimmunology

Article Title: Glutamate promotes CCL2 expression to recruit tumor-associated macrophages by restraining EZH2-mediated histone methylation in hepatocellular carcinoma

doi: 10.1080/2162402X.2025.2497172

Figure Lengend Snippet: EZH2 pathway is essential for glutamate-induced CCL2 expression in HCC.

Article Snippet: For the CCL2-CCR2 studies, mice were treated with CCL2 neutralizing antibody (2 mg/kg, Bio X cell, cat#BE0185) or CCL2 mAb in combination with MK801 intraperitoneally twice a week for 2 weeks.

Techniques: Expressing

Journal: Oncoimmunology

Article Title: Glutamate promotes CCL2 expression to recruit tumor-associated macrophages by restraining EZH2-mediated histone methylation in hepatocellular carcinoma

doi: 10.1080/2162402X.2025.2497172

Figure Lengend Snippet: EZH2-mediated H3K27me3 on the CCL2 promoter to control CCL2 expression.

Article Snippet: For the CCL2-CCR2 studies, mice were treated with CCL2 neutralizing antibody (2 mg/kg, Bio X cell, cat#BE0185) or CCL2 mAb in combination with MK801 intraperitoneally twice a week for 2 weeks.

Techniques: Control, Expressing

Journal: MedComm

Article Title: PDPN+ cancer‐associated fibroblasts enhance gastric cancer angiogenesis via AKT/NF‐κB activation and the CCL2‐ACKR1 axis

doi: 10.1002/mco2.70037

Figure Lengend Snippet: High expression of podoplanin (PDPN), CD31 and chemokine (CC‐motif) ligand 2 (CCL2) in gastric cancer (GC) is associated with poor prognosis. (A) Representative hematoxylin and eosin (H&E) and immunohistochemical images showing PDPN, CD31, and CCL2 protein expression in GC and normal gastric mucus; (B) Semiquantitative analysis of PDPN, CD31, and CCL2 protein expression between GC and normal gastric mucosa; (C–H) Kaplan–Meier survival curves with log‐rank tests depicting disease‐free survival (C, E, G) and overall survival (D, F, H) of GC patients ( n = 400) based on the expression levels of PDPN, CCL2, and intersection of all three proteins in cancer tissues in the in‐house cohort.

Article Snippet: Other antibodies include CD31 (Cat# ab81289, Abcam), CCL2 neutralizing antibody (Cat# MAB679, R&D), human CCL2 (Cat# 300‐04‐5, PeproTech).

Techniques: Expressing, Immunohistochemical staining

Journal: MedComm

Article Title: PDPN+ cancer‐associated fibroblasts enhance gastric cancer angiogenesis via AKT/NF‐κB activation and the CCL2‐ACKR1 axis

doi: 10.1002/mco2.70037

Figure Lengend Snippet: Podoplanin (PDPN)(+) cancer‐associated fibroblasts (CAFs) induce angiogenesis by secreting chemokine (CC‐motif) ligand 2 (CCL2). (A) Cytokine profiles produced by conditioned medium (CM) of PDPN(+) CAFs and PDPN(−) CAFs were examined by RayBio Human Cytokine Antibody Array. The red, blue, and purple frame represents the most significantly overexpressed cytokines. (B) Top 10 cytokines significantly upregulated in CM of PDPN(+) CAFs compared to PDPN(−) CAFs (*** p < 0.001). (C) Enzyme‐linked immunosorbent assay (ELISA) results showing the amount of soluble CCL2 produced by PDPN(+) CAFs, PDPN(−) CAFs, normal fibroblasts (NFs), and human umbilical vein endothelial cells (HUVECs). (D) Uniform manifold approximation and projection (UMAP) of endothelial cells representing three unique cell states, color‐coded by their corresponding cell lineage or subtype. Each dot in the UMAP represents a single cell. (E) Circle plots showing the cellular interactions of CAFs and endothelial cells (ECs) involved in the CCL signaling pathway network in gastric cancer (GC). CAFs and ECs were the core of the cellular interaction network (edge width represents the numbers of interactions and node size represents the abundance of cell populations). (F, G) Wound healing assay (F) and tube formation assay (G) on HUVECs cultured in CM of PDPN(+) CAFs with or without a neutralizing antibody against CCL2 (anti‐CCL2). Scale bar = 100 µm; ** p < 0.01. (H, I) Wound healing assay (H) and tube formation assay (I) on HUVECs treated with or without CCL2 (scale bar = 100 µm; ** p < 0.01). (J) Representative immunofluorescent staining for PDPN, CCL2, and CD31 in clinical GC sample (scale bar = 40 µm). (K) Immunoblot analysis of p‐PI3K, PI3K, p‐AKT, AKT expression in HUVECs treated with CM from PDPN(+) CAFs and CCL2 neutralizing antibody (anti‐CCL2). (L) Immunoblot analysis of p‐PI3K, PI3K, p‐AKT, AKT expression in HUVECs treated with CCL2. (M) Violin plots showing the expression of genes involved in the CCL signaling pathway. CCL2 and CCL11 are primarily expressed in fibroblast subsets, while their potential receptor ACKR1, is predominantly expressed in endothelial cells. (N) Immunoblot analysis of p‐PI3K, PI3K, p‐AKT, AKT expression in HUVECs with indicated treatment.

Article Snippet: Other antibodies include CD31 (Cat# ab81289, Abcam), CCL2 neutralizing antibody (Cat# MAB679, R&D), human CCL2 (Cat# 300‐04‐5, PeproTech).

Techniques: Produced, Ab Array, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Tube Formation Assay, Cell Culture, Staining, Western Blot, Expressing

Journal: MedComm

Article Title: PDPN+ cancer‐associated fibroblasts enhance gastric cancer angiogenesis via AKT/NF‐κB activation and the CCL2‐ACKR1 axis

doi: 10.1002/mco2.70037

Figure Lengend Snippet: Podoplanin (PDPN) regulates NF‐κB activity in cancer‐associated fibroblasts (CAFs) through AKT/IKK signaling, leading to chemokine (CC‐motif) ligand 2 (CCL2) secretion. (A) Heatmap representing significantly dysregulated genes from RNA‐seq analysis of PDPN(+) CAFs and PDPN(−) CAFs ( n = 3). (B) Pathway analysis of differentially expressed genes enriched in PDPN(+) CAFs compared to PDPN(−) CAFs. (C) Immunoblot analysis of nuclear and cytoplasmic p‐P65 and P65 protein expression in PDPN(+) CAFs, PDPN(−) CAFs and normal fibroblasts (NFs). (D) Representative immunofluorescent staining of p‐P65 in PDPN(+) CAFs, PDPN(−) CAFs, and NFs (scale bar = 25 µm). (E) Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) results showing the differential mRNA expression levels of CCL2 in PDPN(+) CAFs transfected with P65 siRNA or control siRNA ( n = 3; ** p < 0.01). (F) Enzyme‐linked immunosorbent assay (ELISA) results showing the amount of soluble CCL2 in conditioned medium (CM) from PDPN(+) CAFs transfected with P65 siRNA or control siRNA (** p < 0.01). (G, H) Wound healing assay (G) and tube formation assay (H) on human umbilical vein endothelial cells (HUVECs) treated with CM from PDPN(+) CAFs transfected with P65 siRNA or control siRNA (scale bar = 100 µm; ** p < 0.01). (I) Chromatin immunoprecipitation (ChIP) assay was performed to verify P65 binding to the CCL2 promoter. CCL2 promoter segments were quantified using qRT‐PCR, with results normalized against IgG. Data are presented as the mean ± SD from three independent experiments is presented (*** p < 0.001). (J) Top: Schematic representation of the CCL2 reporter construct. The consensus P65 binding sequences are marked as with blue box. The consensus sequence and the putative P65 binding site sequences are shown. Bottom: Effects of ectopic expression of P65 siRNA on wild type (WT) and mutant (MUT) CCL2 promoter reporter activity (** p < 0.01). (K) Effects of ectopic expression of P65 siRNA (siP65#1), AKT inhibitors perifosine (10 µmol/L), NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC, 0.05 µmol/L) on CCL2 promoter reporter activity (** p < 0.01). (L) Immunoblot analysis of AKT/IKK pathway in PDPN(+) CAFs, PDPN(−) CAFs and NFs. (M) Immunoblot analysis of AKT/IKK/NF‐κB pathway and nuclear/cytoplasmic distribution of P65 in PDPN(+) CAFs treated with or without perifosine (10 µmol/L) and LY294002 (20 µmol/L) for 24 h.

Article Snippet: Other antibodies include CD31 (Cat# ab81289, Abcam), CCL2 neutralizing antibody (Cat# MAB679, R&D), human CCL2 (Cat# 300‐04‐5, PeproTech).

Techniques: Activity Assay, RNA Sequencing, Western Blot, Expressing, Staining, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Control, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Tube Formation Assay, Chromatin Immunoprecipitation, Binding Assay, Construct, Sequencing, Mutagenesis

Journal: MedComm

Article Title: PDPN+ cancer‐associated fibroblasts enhance gastric cancer angiogenesis via AKT/NF‐κB activation and the CCL2‐ACKR1 axis

doi: 10.1002/mco2.70037

Figure Lengend Snippet: Targeting podoplanin (PDPN)(+) cancer‐associated fibroblasts (CAFs) restrains angiogenesis via inhibiting AKT/NF‐κB signaling. (A) Schematic diagram of in vivo animal experiment. (B) Tumor image (left) and tumor growth curve (right) of xenografted mice inoculated with HGC27 cells and PDPN(+) CAFs pretreated with or without perifosine (10 µmol/L) and Bay117082 (10 µmol/L; *** p < 0.001). (C) Representative images of hematoxylin and eosin (H&E) and immunofluorescent staining for CD31 as indicated in the xenografted tumors (scale bar = 40 µm; * p < 0.05). (D) Schematic view of the proposed mechanism. A schematic diagram illustrating the proposed mechanism by which PDPN(+) CAFs‐derived chemokine (CC‐motif) ligand 2 (CCL2) promotes angiogenesis in gastric cancer (GC). The Akt/NF‐κB pathway was significantly activated in PDPN(+) CAFs. P65 directly bind to the CCL2 promoter, thereby increasing the CCL2 transcription and secretion in CAFs; CCL2, which was transcriptional activated by P65 in PDPN(+) CAFs, sustains tumor angiogenesis by interacting with ACKR1 and activating PI3K/AKT signaling in endothelial cells. The scheme was drawn by biorender ( https://biorender.com/ ).

Article Snippet: Other antibodies include CD31 (Cat# ab81289, Abcam), CCL2 neutralizing antibody (Cat# MAB679, R&D), human CCL2 (Cat# 300‐04‐5, PeproTech).

Techniques: In Vivo, Staining, Derivative Assay

Journal: Frontiers in Immunology

Article Title: ICAM1 + gingival fibroblasts modulate periodontal inflammation to mitigate bone loss

doi: 10.3389/fimmu.2024.1484483

Figure Lengend Snippet: Inflammatory ICAM1 + fibroblasts expand in a murine model of ligature-induced periodontitis. (A) Flow cytometry gating strategy for analysis of lineage-negative (CD45 - CD31 - EpCam - Ter119 - ) pericytes, gingival fibroblasts and ICAM1 + fibroblasts in non-ligated control (NL) and ligature induced periodontitis (LIP) group. (B) Quantification of percent fibroblasts (Lin - PDGFRA + ) and pericytes (Lin - CD146 + ) normalized by Lin - mesenchymal cell numbers in NL and LIP groups. Each dot represents one mouse as a split-mouth design. (C) Quantification of percent ICAM1 + fibroblasts (Lin - PDGFRA + ICAM1 + ) normalized by total fibroblast numbers. Each dot represents one mouse as a split-mouth design. (D) Representative immunofluorescent images of NL and LIP paraffin sections stained with antibodies specific against ICAM1 (red) and PDGFRA (green). Arrows point to ICAM1 + PDGFRA + cells. Scale bar, 100μm; inset scale bar, 10μm. (E) Quantification of ICAM1 fibroblast numbers normalized by lamina propria area (mm 2 ) comparing NL and LIP groups from the immunofluorescence experiments. (F) Left, representative immunocytochemistry images of primary gingival fibroblasts stained with ICAM1 antibody comparing control versus stimulated groups. Lipopolysaccharide from P. gingivalis (LPS, 1 ug/ml) and tumor necrosis factor alpha (TNF, 10 ng/ml) were used for stimulation. Scale bar, 20 μm. Right, quantification of ICAM1 + fibroblast numbers normalized by total fibroblast cells comparing control and LPS+TNF group. (G) Schematic diagram of qPCR for fibroblast-derived cytokines comparing FACS-sorted ICAM1 - and ICAM1 + fibroblasts in human and mouse models of periodontitis. (H, I) Quantification of relative mRNA expression by qPCR for CXCL13, CXCL1, CXCL2, CCL19, and CCL2 comparing sorted ICAM1 - and ICAM1 + fibroblasts. (H) Gingival tissue specimens from N=8 periodontitis patients; each dot represents individual patient. (G) Gingival tissues harvested from mice with LIP; each dot represents pooled samples from 2-3 mice for a total of 4 data points (N=10 mice). Data represent mean ± SEM. Welch’s t-test (B–H) and Mann-Whitney U test (I) comparing control vs. experimental group; *p<0.05, **p<0.01, ***p<0.001, ns, not significant.

Article Snippet: BMMs were then stimulated with low-dose LPS (10 ng/ml) for 24h to induce macrophage phagocytotic phenotype, followed by incubation in conditioned media from the ICAM1 + enriched or control fibroblasts, with or without neutralizing anti-CCL2 monoclonal antibody (20 μg/ml, eBioscience, 16-7096-81).

Techniques: Flow Cytometry, Control, Staining, Immunofluorescence, Immunocytochemistry, Derivative Assay, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: ICAM1 + gingival fibroblasts modulate periodontal inflammation to mitigate bone loss

doi: 10.3389/fimmu.2024.1484483

Figure Lengend Snippet: (A) Left, representative flow cytometry histogram of CCL2 signal in 7d ligated control and experimental mice. Middle and right, quantification of percent CCL2 + ICAM1 + fibroblasts (Lin - PDGFRA + ) and CCL2 + pericytes (Lin - CD146 + ) normalized by lineage-negative mesenchymal cell numbers. (B) Representative flow cytometry histogram of ICAM1 expression in fibroblasts pre-gated for CCL2 + signal in control or ICAM1 + oral fibroblast-enriched conditions (LPS + TNF) treated with or without BMS-345541 in vitro. (C) Left, quantification of ICAM1 + CCL2 + fibroblasts cell counts by flow cytometry analysis. Right, ELISA analysis of CCL2 concentration in the supernatant of cultured control or ICAM1 + enriched fibroblast conditions with or without BMS-345541. (D) Top, representative gating strategy for CCL2 + cell phenotyping by flow cytometry using tissues collected from CCL2 mCherry reporter mice that had ligature placed for 7 days. Bottom right, quantification of CCL2 + fibroblasts (CD45 - EpCAM - PDGFRA + ), leukocytes (CD45 + ), endothelial cells (CD31 + ), epithelial cells (EpCAM + ), and pericytes (CD31 - CD146 + ) normalized by total CCL2 + cells. Each dot represents one mouse (N=4). (E) Top, representative immunofluorescent images of non-ligated control (NL) and ligature induced periodontitis (LIP) from CCL2 mCherry reporter mice. Paraffin sections were stained with antibodies specific against PDGFRA (green) and red fluorescent protein (red), and immunopositivity in the lamina propria and periodontal ligament space (PDL) was examined. Scale bar, 20μm.Bottom, quantification of percent CCL2 + fibroblasts (CCL2 + PDFGRA + ) normalized by total nucleated cells in field of view. N=3, split mouth design. (F) Schematic diagram of in vitro phagocytosis assay using conditioned media from ICAM1 + enriched oral fibroblast culture and primary bone marrow-derived macrophages. (G) Top, flow cytometry gating strategy for identification of double positive F4/80 + fluorescence beads + from in vitro phagocytosis assay. Bottom, representative flow cytometry histogram of fluorescence beads signals showing three distinct peaks from control, conditioned media (CM), and CM + anti-CCL2 neutralization groups. (H) Quantification of fluorescence beads + F4/80 + macrophage numbers per 10 4 events. Left, number of beads + F4/80 + with a first peak (one bead) in the histogram; right, number of beads + F4/80 + with a second or third peak (two or three beads phagocytosed). N=3 each. All in vitro experiments were repeated independently twice. Data represents mean ± SEM. For (A) , one-way ANOVA test followed by pairwise t-test’s with Šidák’s correction was performed. For (C) Brown Forsythe ANOVA test with Dunnett’s T3 Multiple comparison test; ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: BMMs were then stimulated with low-dose LPS (10 ng/ml) for 24h to induce macrophage phagocytotic phenotype, followed by incubation in conditioned media from the ICAM1 + enriched or control fibroblasts, with or without neutralizing anti-CCL2 monoclonal antibody (20 μg/ml, eBioscience, 16-7096-81).

Techniques: Flow Cytometry, Control, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Staining, Phagocytosis Assay, Derivative Assay, Fluorescence, Neutralization, Comparison