recombinant human ccl28  (PeproTech)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: CCL28 expression is upregulated after anti-angiogenesis therapy by hypoxia-sensitive transcription factor CEBPB in lung adenocarcinoma

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Tumor-derived CCL28 recruits pericytes to promote vascular normalization in the tumor microenvironment

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Tumor-derived CCL28 promotes the expression of angiopoietin-1 via CCR3 in pericytes

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Retinoic acid signaling is activated by CCL28 in pericytes through CCR3 A , Volcano plot of changes in metabolic pathways after CCL28 stimulation. B Volcano plot of the enrichment of gene expression after CCL28 stimulation. C Diagram of the metabolic conversion process in the retinoic acid metabolic signaling pathway. D and E Expression of RDH13 and DHRS11 detected by qPCR and western blot with or without exogenous supplement of CCL28. F Correlation of expression of CCL28 with RDH13 in lung adenocarcinoma. G The protein level of DHRS11 and RDH13 stimulated with or without CCL28 and CCR3 neutralizing antibody in pericytes (left) and gray value was calculated(right). H Knockdown efficiency of RDH13 was confirmed by qPCR. I and J Relative expression of RXRα and ANGPT1 after knockdown of RDH13 with or without stimulation of CCL28. K Representative immunofluorescence images of PAN-CK, NG2, CCL28 with DHRS11 or RDH13 or Angiopoietin-1 on biopsy tissues from lung cancer patients (left panel). Scale bar = 100 μm. The correlation between the expression of CCL28 and the levels of DHRS11, RDH13, and angiopoietin-1 (right panel). Data with error bars are shown as mean ± SEM. Each symbol represents data from a replicate. Each panel is a representative experiment of at least three independent biological replicates. *, **, *** represent p < 0.05, p < 0.01 and p < 0.001, respectively. Abbreviation: MFI, Mean fluorescence intensity

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Expressing, Western Blot, Knockdown, Immunofluorescence, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Both CCL28 and retinoic acid could promote vascular normalization in vivo

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: In Vivo

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: CCL28 is involved in bevacizumab-mediated vascular normalization

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: A schematic diagram of tumor microenvironment modulation effects of CCL28

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Invasion of Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β (mean ± SEM, n = 3). *P < 0.05 vs. cells without CCL28 and TGF-β; #P < 0.05, ##P < 0.005 vs. TGF-β–only–treated cells by 1-way ANOVA with multiple-comparisons test. (B) Invasion of Ca9.22 and YD10B OSCC cells with CCL28 and/or TGF-β into the CAMs of fertilized eggs (mean ± SEM, n = 3). Representative images of CAM. Scale bars: 100 μm. Cells invaded into the mesoderm layer of CAMs are quantified by the mean fluorescence. *P < 0.05, **P < 0.01 vs. cells without CCL28 and TGF-β; #P < 0.05, ##P < 0.001 vs. TGF-β–only–treated cells by 1-way ANOVA with multiple-comparisons test. (C) Expression levels and cellular localization of E-cadherin and β-catenin in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. Representative immunofluorescence images. Scale bars: 100 μm. (D) Expression levels of E-cadherin, β-catenin, and EMT-regulating transcription factors in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. (E) Cytosolic and nuclear β-catenin levels in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. (D and E) Representative Western blot images.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques: Fluorescence, Expressing, Immunofluorescence, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Invasion of CCL28-knockdown OSCC cells. (B) Invasion of CCR3-knockdown OSCC cells. (C) Invasion of CCR10-knockdown OSCC cells. (A–C) OSCC cells were transduced with lentiviral particles with control shRNAs or 3 different shRNAs targeting CCL28, CCR10, or CCR3. Knockdown of CCL28, CCR10, or CCR3 in transduced cells was confirmed by Western blotting (top panels). Cell invasion is quantified as the number of invaded cells per field (mean ± SEM, n = 3). *P < 0.05, **P < 0.005 vs. control shRNA–transfected cells without CCL28; #P < 0.05, ##P < 0.01 vs. CCL28-, CCR3-, or CCR10-specific shRNA–transfected cells without CCL28 by 1-way ANOVA with multiple-comparisons test. (D) Invasion of CCL28- or CCR10-knockdown OSCC cells labeled with CFDA-SE and then suspended in a DMEM/Matrigel (4:1) mixture on the CAMs of fertilized eggs (mean ± SEM, n = 3). Representative images of CAM. Scale bars: 100 μm. Cells invaded into the mesoderm layer are quantified by the mean fluorescence. *P < 0.05 versus control shRNA–transfected cells without CCL28; #P < 0.01 vs. CCL28- or CCR10-knockdown cells without CCL28 by 1-way ANOVA with multiple-comparisons test. (E) CCL28, CCR3, or CCR10 mRNA levels in normal and HNSCC tissues. The data were obtained from the TCGA database. Box plots show the median and interquartile range. *P < 0.0001 vs. normal tissue by 2-tailed Student’s t test. (F) Kaplan-Meier survival curves for HNSCC patients with high or low expression of CCL28, CCR3, or CCR10 mRNA by the log-rank test.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques: Transduction, Western Blot, shRNA, Transfection, Labeling, Fluorescence, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Representative pathway reporter array (n = 2) for wild-type and CCR10-knockdown (KD) OSCC cells in the absence or presence of CCL28 (20 ng/mL). Reporter gene activities in CCL28-treated cells were normalized by those in untreated cells and represented as fold changes. (B) Correlations between CCL28 mRNA expression and RARβ mRNA expression in patients with HNSCC by Pearson’s correlation analysis. Scatter plots represent normalized RSEM values for each gene. (C) RARβ and RARβ2 expression in response to CCL28 treatment (20 pg/mL) in Ca9.22, YD10B, HSC2, or HSC3 OSCC cells. (D) RARβ and RARβ2 expression in CCL28-overexpressing or CCL28-knockdown Ca9.22 or YD10B OSCC cells. (E) RARβ expression in response to CCL28 treatment (20 pg/mL) in CCR3- or CCR10-downregulated Ca9.22 or YD10B OSCC cells. (C–E) Representative Western blot images. (F) Invasion of OSCC cells treated with the RARβ-selective antagonist LE135 or the inverse pan-RAR agonist BMS493 in the presence of CCL28 (20 pg/mL) (mean ± SEM, n = 3). *P < 0.05 and **P < 0.005 versus CCL28-untreated cells; #P < 0.05 and ##P < 0.01 versus CCL28-only-treated cells by 1-way ANOVA with multiple-comparisons test.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques: Expressing, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) RARβ and RARβ2 expression levels in OSCC cells treated with CCL28 (20 pg/mL) and/or the selective RARα antagonist ER50891 (ER). (B) Invasion of OSCC cells treated with CCL28 (20 pg/mL) and/or the selective RARα antagonist ER50891 (ER) (mean ± SEM, n = 3). *P < 0.001 versus CCL28-untreated control cells; #P < 0.005 and ##P < 0.001 versus CCL28-only-treated cells by 1-way ANOVA with multiple-comparisons test. (C) Interaction between RARα and HDACs or DNMT in OSCC cells treated with CCL28 (20 pg/mL). Immune complexes were obtained using a Pierce Co-IP kit. (A and C) Representative Western blot images. (D) Acetylated histone H3 levels and HDAC1 interaction at the RARB promoter region of OSCC cells treated with CCL28 (20 pg/mL). Histone modification (H3K9ac) and HDAC1 binding were analyzed by ChIP-qPCR. Data are presented as the percentage of the total chromatin input (% input), and graphs are representative.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Modification, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) RANKL and OPG levels secreted by CCL28-treated OSCC cells into the culture media, and the RANKL/OPG ratio (mean ± SEM, n = 3). *P < 0.05 vs. CCL28-untreated cells by 2-tailed Student’s t test. (B) RANKL levels secreted by OSCC cells treated with the selective RARα antagonist ER50891 or the RARβ antagonist LE135 in the presence of CCL28 (mean ± SEM, n = 3). *P < 0.05 versus CCL28-untreated cells; #P < 0.05 versus CCL28-only-treated cells by 1-way ANOVA with multiple comparisons test. (C) RANKL and OPG levels secreted by CCL28-treated osteoblasts into the culture media, and the RANKL/OPG ratio (mean ± SEM, n = 3). *P < 0.05 and **P < 0.01 versus CCL28-untreated cells by 1-way ANOVA with multiple comparisons test. (D) Secreted levels of RANKL and OPG by CCL28-treated osteoblasts in the presence of conditioned media (CM) from OSCC cell lines, and the RANKL/OPG ratio (mean ± SEM, n = 3). #P < 0.05 and ##P < 0.01 versus control cells without CM; *P < 0.05 versus CM-only-treated cells by 1-way ANOVA with multiple-comparisons test. (E) Osteoclast formation in CCL28-treated BMMs in the presence of RANKL (mean ± SEM, n = 3). Representative images at ×100 original magnification. *P < 0.05 versus RANKL-only-treated cells by 1-way ANOVA with multiple comparisons test.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: CCL28 was intraperitoneally administered to mice subcutaneously injected with Ca9.22 OSCC cells in the calvaria (n = 5 for control and n = 10 for experimental groups). (A) Tumor size (mean ± SEM). #P < 0.001 versus vehicle-treated mice by 1-way ANOVA with multiple comparisons test. (B) Representative CT 3D images of calvarial osteolytic lesions. (C) Bone morphometric parameters BV/TV and BS/TV (mean ± SEM). (D) Serum levels of bone turnover markers (mean ± SEM). (E) Representative images of H&E and TRAP staining in calvarial tissue sections. Scale bars: 100 μm. (F) Oc.S/BS determined from TRAP staining as the percentage of bone surface in contact with osteoclasts (mean ± SEM). (C, D, and F) #P < 0.05, ##P < 0.01, and ###P < 0.005 versus control mice; *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test. (G) Ki67, CD31, and RAR expression levels in calvarial tumor tissues of OSCC-injected mice. Left panel: Representative images of immunohistochemically stained tumor tissues. Scale bars: 100 μm. Graph shows quantified data. *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple-comparisons test.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques: Injection, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: CCL28 was intraperitoneally administered to mice injected with YD10B OSCC cells into the bone marrow of the right tibia (n = 5 for control and n = 7 for experimental groups). (A) Representative CT 3D images of osteolytic lesions in the tibia. (B) Bone morphometric parameters (mean ± SEM). (C) Serum levels of bone turnover markers (mean ± SEM). (D) Representative images of H&E and TRAP staining in tibial tissue sections. Scale bars: 100 μm. (E) Tumor area determined from H&E staining as the percentage of the total tumor area per tissue area. (F) Oc.S/BS determined from TRAP staining as the percentage of bone surface in contact with osteoclasts (mean ± SEM). (B, C, E, and F) #P < 0.05, ##P < 0.01, and ###P < 0.005 versus control mice; *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test. (G) Ki67, CD31, and RARβ expression levels in tibial tumor tissues of OSCC-injected mice. Left panel: Representative images of immunohistochemically stained tumor tissues. Scale bars: 100 μm. Right panel: Ki67-positive cells, CD31-positive vessels, and RARβ-positive cells were counted in tumor tissues. *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques: Injection, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Representative images of IHC staining of CCL28, CCR3, CCR10, and RARβ in normal oral mucosa and OSCC tissues. Scale bars: 100 μm. Magnified images of the boxed area are shown in the insets. Scale bars: 20 μm. (B) Frequency of histoscores in normal oral mucosa and OSCC tissues. (C) Kaplan-Meier survival curve of patients with OSCC stratified based on CCL28, CCR3, CCR10, or RARβ expression by the log-rank test.

Article Snippet: Recombinant human CCL28 and TGF-β were obtained from PeproTech.

Techniques: Immunohistochemistry, Expressing

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: CCR10-transfected HepG2 and LO2 cells were treated with either the CCR10 agonist-ligand CCL28 or the Akt inhibitor A6730. a Activation of the CCL28-CCR10 axis by CCL28 significantly increased Akt phosphorylation, PCNA protein expression, and b relative cell proliferation in both cell lines, while Akt inhibition produced the opposite effects. Relative cell proliferation is defined as the fold-change in proliferation relative to the untreated parent cell line. * P < 0.05 vs. CCR10 group. All values are reported as means ± standard errors of the mean (SEMs)

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Transfection, Activation Assay, Phospho-proteomics, Expressing, Inhibition, Produced

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: Following short-term DEN-induced inflammation (10 days after i.p. DEN injection), ( a ) Western blotting analysis showed significantly enhanced TNF protein expression, CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Knocking-out CCR10 significantly opposed these inflammation-induced effects but did not significantly affect TNF or PI3K protein expression.* P < 0.05 vs. vehicle WT group, † P < 0.05 vs. DEN-treated WT group. b Western blotting analysis of CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression in murine liver tissue 6 h after intraperitoneal (i.p.) injection of TNF, which produced significant increases in CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Pretreatment with the CCR10 agonist-ligand CCL28 significantly increased Akt phosphorylation and PCNA expression levels, while pretreatment with the Akt inhibitor A6730 produced the opposite effects. Neither CCL28 nor A6730 had any significant effect upon CCR10 or PI3K expression. * P < 0.05 vs. vehicle group, † P < 0.05 vs. TNF group. All values are reported as means ± standard errors of the mean (SEMs). n = 12 mice in each group

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Injection, Western Blot, Expressing, Phospho-proteomics, Produced

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A–B ) cDNA was isolated from a panel of human pancreatic cancer cell lines, Panc1, MiaPaCa2, Capan2, Capan1, HPAFII, and Hs766t, and screened by RT-PCR for expression of CCR (A), CXCR, CX 3 CR, or XCR (B) family of receptors. C–D ) The same panel of human cell lines was probed for expression of the known ligands for CCR10 (C) and CXCR6 (D). E ) RT-PCR analysis of patient-derived pancreatic cells (MCW PDAC cell lines) as well as a human pancreatic epithelial nestin-expressing (HPNE) confirmed CXCR6-CXCL16 and CCR10-CCL28 transcript expression. F ) Pancreatic tumor cells derived from the KPC murine model exhibited varying levels of the ligands and receptors. GAPDH and actin were analyzed as loading controls. Regions of the MBL and NRAMP genes were assessed as genomic DNA controls. Positive control was RNA from PBMCs.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Positive Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Normal pancreatic tissue was sectioned and processed for histopathologic analysis for CCL28, CCR10 and CK19. The arrowhead denotes the epithelial cell lining. B) Tissue from pancreatic tumor was sectioned and processed for immunohistochemical staining with antibodies specific for CCL28, CCR10, CK19, α-SMA as well as H&E, Masson’s trichrome (3C) or Movat’s pentachrome (5C). ‘T’ denotes the tumor cells and ‘S’ denotes the stromal compartments of the tissue section as defined by CK19, α-SMA and trichrome staining. C–D ) Representative stained tissue was scored by an investigator blinded to the tissue or antibody source for staining intensity. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Tissue staining shown in A and B representative of tissues sections from 14 normal, 12 PanIN, and 12 PDAC patients, based on clinical diagnosis and examination by a board-certified pathologist.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Biomarker Discovery

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A) Sections of pancreatitis tissue were processed for immunostaining of CCR10 and CCL28, CXCR6, and CXCL16. B–C) . Scoring for the number of ductal (CK19+) or stromal (SMA+) regions for stain of CXCL16, CXCR6, CCL28 or CCR10. Gray line is the mean staining intensity of normal pancreatic tissue shown in and included as a reference. D ) Parallel tissue sections were processed for immunohistochemistry for CXCL12, CXCR7, CXCR4 and CK19. E ) Quantitative staining intensity of CXCL12, CXCR7 and CXCR4 in CK19+ cells. *** = P ≤ 0.001, **** = P ≤ 0.0001. Tissue staining shown in A and D are representative of tissue specimens from 5–9 individual patients clinically and pathologically diagnosed with pancreatitis.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunostaining, Staining, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Sandwich ELISA revealed that the panel of tissue culture pancreatic cancer cells secrete CCL28 when stimulated with 50 ng/mL IFNγ. Levels of CCL28 were undetectable in HPSCs treated with IFNγ. B ) Flow cytometric analysis of HPSCs revealed the cell surface expression of CCR10 (top panel). The mean fluorescence intensity (MFI) was determined for cells stained with the isotype control primary antibody (light gray) and anti-CCR10 antibody (dark gray). C ) CCL28 stimulates chemotaxis. HPSCs were plated in the top well of a transwell insert in serum free media. The bottom well contained media (serum free; SF or full growth; FG). Recombinant CCL28 [30 nM] was added to the bottom or top well as indicated. After 4h, inserts were swabbed, stained with DAPI and enumerated. D ) CCL28-mediated HPSC migration is dose dependent. HPSCs were plated in the top well of a transwell insert. The bottom well contained either full growth media (+; positive control) or serum-free media that either lacked stimulant (-; negative control), or increasing concentrations of recombinant CCL28. E ) HPSC migration is mitigated by an anti-CCL28 neutralizing antibody. The transwell migration setup as above was modified to include the following conditions: no stimulation (NS; negative control), 10 ng/mL TGF-β (positive control) or 30 nM recombinant CCL28. Neutralizing antibody to CCL28 was added to the bottom well for 30 minutes prior to incubation with the cells. F ) CCL28-mediated directional migration of HPSCs is through a G-coupled protein receptor pathway. HPSCs were pretreated with pertussis toxin (PTX) or vehicle, prior to CCL28 stimulation. G) Transcript expression of CCR10 in HPSC cells. RNA was isolated and used as a template for RT-PCR analysis using CCR10 or GAPDH as a loading control. H) CCL28 stimulates chemotactic migration of patient-derived HSPC2 cells. HPSC2 cells were added to a transwell insert and placed in a well containing either serum free medium in the absence (NS) or presence of CCL28 (CCL28) or full growth medium (FG) as a positive control (FG). * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Sandwich ELISA, Expressing, Fluorescence, Staining, Control, Chemotaxis Assay, Recombinant, Migration, Positive Control, Negative Control, Modification, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Three independent HPSC lines were treated with 30 nM CCL28, left untreated (NS), treated with 10 ng/mL TGF-β (C+), treated with SB-431542 [25 µM], a TGF-β1R inhibitor (C-) or treated with vehicle controls (V). A) Representative immunofluorescence images show digital intensity “heat maps” of detected αSMA, corresponding to intensity color bar scale. Scale bars represent 50 µm. B) Semi-quantitative imaging analysis of the mean integrated intensity (Integ Int) of αSMA detection normalized for each HPSC line (HPSC, 2, 3) using the vehicle (V) set as 1 arbitrary unit. HPSC: #, P = 0.0179. ***, P ≤ 0.0002. HPSC2: #, P = 0.0144; ****, P <0.0001; HPSC3, *****, P < 0.0001. C) Representative images of HPSC-derived ECMs produced under experimental conditions as in A and analyzed using Orientation-J plugin of Image-J software. Color tones were normalized using hue values for common mode angle (cyan/green boarded color) visualization as represented on the orientation bar. D) Box and whisker plot of mean percent of fibers distributed within 15° angles from the mode corresponding to the indicated experimental conditions. HPSC: #, P = 0.0304; *, P = 0.028; HPSC2: *, P = 0.0117; HPSC3: C + versus Vs, *, P = 0.0101. No substantial ECM production was obtained from HPSC3 under TGF-β inhibition and was designated as not available (NA).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunofluorescence, Imaging, Derivative Assay, Produced, Software, Whisker Assay, Inhibition

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Schematic representation of the experimental plan ( A ). Briefly, PDAC epithelial cells were seeded to the bottom well of a transwell dish (1) then stimulated with 50 ng/mL IFNγ to elicit chemokine secretion. HPSCs were plated onto the top chamber of the transwell insert (2) while CCL28 neutralizing antibody was added to the bottom chamber. The insert with HPSCs was added (3) and incubated for 4h prior to (4) fixing, DAPI-staining and cell counting. Stimulation of Panc1 ( B–C ) and MiaPaCa2 ( D–E ) cells by IFNγ resulted in directional migration of HPSCs that was inhibited by the neutralizing CCL28 antibody. HPSC migrate towards PDAC tumor cells and not non-transformed epithelial HPNE cells ( F–G ). Representative images ( C, E ) of 5 independent biological replicates are presented. * = P ≤ 0.05. ** = P ≤ 0.01.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Incubation, Staining, Cell Counting, Migration, Transformation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Normal pancreatic duct epithelial cells and quiescent stromal fibroblasts almost no ligand CCL28 (purple circles) and basal levels of the receptor CCR10 (left panel). In inflammation, such as in pancreatitis, CCL28 expression is increased by cytokines such as IFNγ, which also participate in the activation of stellate cells into cancer-associated fibroblasts. CCL28 directs the migration of stellate cells toward the epithelium (middle panel). CCL28 produced by transformed ductal cells direct the sustained migration of activated stellate cells into the remodeling tumor microenvironment (right panel).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Expressing, Activation Assay, Migration, Produced, Transformation Assay

Journal: Scientific Reports

Article Title: Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells

doi: 10.1038/srep27152

Figure Lengend Snippet: ( A ) Expression changes of all the chemokines in lung adenocarcinoma cells under hypoxic condition. Lung adenocarcinoma cells, A549 and SPC-A1, were cultured on plates (2D) or in matrigel (3D) under hypoxic condition (1% O 2 ) for 24 hours. Cells cultured under normoxia (20% O 2 ) were set as control. Several classical hypoxia induced genes, such as CA9 , GLUT1 and VEGFA , were significantly up-regulated. Of all chemokines, CCL28 was the only one that significantly up-regulated and there was slightly up-regulation of CXCL8. ( B ) Confirming the results from microarray by real time RT-PCR. Expression of CCL28 was significantly up-regulated in both A549 and SPC-A1 cells when cultured in hypoxia chamber (1% O 2 ). ( C ) fresh lung adenocarcinoma tumor samples(n = 4), scissored into pieces with a diameter less than 2 mm, were cultured under two different oxygen concentrations, 1 and 20%. RT-PCR was applied to detect the expression of CCL28. Expression of CCL28 was significantly up-regulated in two lung adenocarcinoma tumors (P1 and P2) when cultured in hypoxia chamber (1% O 2 ). The bands were cropped from the original gel images in . ( D,E ) expression of CCL28 in lung adenocarcinoma tumors. Distributions of CCL28 were consistent with that of HIF-1alpha, a standard hypoxia marker (D, representative photos from two patients, P1 and P2). And the intensity of CCL28 expression was also correlated with that of HIF-1alpha expression ( E ), n = 15 and p < 0.05. ( F ) serum concentration of CCL28 was much higher in lung adenocarcinoma patients, comparing with that in healthy donors (p < 0.001). ( G ) as a classical hypoxia induced gene, we further examined the serum concentration of VEGFA in lung adenocarcinoma patients. There was a significant correlation between the serum concentration of CCL28 and VEGFA (n = 22, p = 0.009). Data were expressed as mean ± SEM. LAC, lung adenocarcinoma. **p < 0.01; scale bar, 50 μm.

Article Snippet: Recombinant human CCL28 and recombinant human VEGFA (10 ng/ml, R&D Systems, USA) were added into culture medium.

Techniques: Expressing, Cell Culture, Microarray, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Marker, Concentration Assay

Journal: Scientific Reports

Article Title: Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells

doi: 10.1038/srep27152

Figure Lengend Snippet: (A,B ) human pulmonary microvascular endothelial cells (HPMEC) were seeded in 24-well plate pre-coated with matrigel (5 × 10 4 /well). The total tube length was analyzed by ImageJ. CCL28 (2000 ng/ml) and VEGFA (10 ng/ml) significantly increased the formed tube length of HPMEC. In addition, the effect of CCL28 on tube formation was neutralized by antibody against CCR3 (CCR3 Ab). ( C,D ) wound healing assay of HUVEC indicated that CCL28 increased migration of endothelial cells. ( E,F ) when seeded with a low number (1 × 10 4 cells/well) on matrigel, HPMEC cells could proliferate and form cell clones. The number of cell clones was significantly increased by CCL28 (p < 0.01), as well as VEGFA, and this effect of CCL28 could also be neutralized by neutralizing CCR3 antibody. NC, negative control; NS, no significant. Data were expressed as mean ± SEM. *p < 0.05; **p < 0.01; scale bar, 50 μm.

Article Snippet: Recombinant human CCL28 and recombinant human VEGFA (10 ng/ml, R&D Systems, USA) were added into culture medium.

Techniques: Wound Healing Assay, Migration, Clone Assay, Negative Control

Journal: Scientific Reports

Article Title: Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells

doi: 10.1038/srep27152

Figure Lengend Snippet: ( A,B ), matrigel was mixed with CCL28 (2000 ng/ml) and implanted into T cell deficient BALB/c nude mice (CByJ.Cg- Foxn1 nu /J, female, 4–6 weeks old) subcutaneously. CCL28 induced more angiogenesis in matrigel, which was analyzed by immunofluorescence and the vascular area was calculated ( B–D ), effects of CCL28 on angiogenesis analyzed by Chick chorioallantoic membrane (CAM) assay. As a positive and negative control, VEGFA (10 ng/ml) and cisplatin (DDP, 2 μg/ml) could significantly induce and inhibit angiogenesis on chick chorioallantoic membrane, respectively (p < 0.01). While CCL28 (2000 ng/ml) also induced more angiogenesis on chick chorioallantoic membrane. Furthermore, the effect of CCL28 on angiogenesis was neutralized by antibody against CCR3 (CCR3 Ab). Black circles indicated the positions where the Gelatin Sponges were placed. Vascular areas were analyzed on the whole taken photos. ( E–G ), lung adenocarcinoma cell lines, A549 and SPC-A1, with stable over-expression of CCL28 were screened out and implanted subcutaneously in BALB/c nude mice. Compared with control, tumors with CCL28 high expression had a significant higher growth rate ( F , p < 0.05). In addition, the tumor microvascular density was much higher in CCL28 high expression tumors ( E and G , p < 0.05). ( H–J ), A549 and SPC-A1 cells with CCL28 expression knockdown by RNA interfering had a lower rate of tumor formation in BALB/c nude mice ( H ). And there was a lower level of tumor microvascular density in CCL28 knockdown tumors ( I and J , p < 0.05). The vascular areas or densities were analyzed by ImageJ. NC, negative control; NS, no significant. Data were expressed as mean ± SEM. *p < 0.05; **p < 0.01; scale bar, 50 μm.

Article Snippet: Recombinant human CCL28 and recombinant human VEGFA (10 ng/ml, R&D Systems, USA) were added into culture medium.

Techniques: Immunofluorescence, Membrane, Chick Chorioallantoic Membrane Assay, Negative Control, Over Expression, Expressing

Journal: Scientific Reports

Article Title: Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells

doi: 10.1038/srep27152

Figure Lengend Snippet: Phosphorylation Antibody Arrays were applied to detect the changes of phosphorylation of signaling proteins in HPMEC cells treated by CCL28 (2000 ng/ml) and VEGFA (10 ng/ml), with normal saline as control. The cut-off value for the phosphorylation was set up as 1.5. ( A ) heat map represented the results of VEGFR Pathway Phosphorylation Antibody Array. As a positive control, VEGFA (10 ng/ml) activated the whole VEGFR signaling pathway ( A , left panel). CCL28 also induced phosphorylation of twenty signaling proteins ( A , right panel). ( B ) scanned photos of Phosphorylation Antibody Arrays, consistent with twenty phosphorylated signaling proteins in A (right panel) as indicated by dashed lines. ( C ) the phosphorylated proteins induced by CCL28 could be grouped into three signaling pathways, including PI3K-Akt, MAPK and G protein-coupled receptor signaling pathways. ( D,E ) Verification of the results of Phosphorylation Antibody Arrays. Western Blot was applied to detect phosphorylation of p38 MAPK (Tyr322), Akt (Ser473), eNOS(Ser1177) and PKCα/β II(Thr638/641) in HPMEC cells treated with CCL28 (2000 ng/ml). The bands were cropped from the original blot images in . p38 MAPK, Akt, eNOS and PKCα/β II were significantly phosphorylated in HPMEC cells after treatment of CCL28. And this effect was neutralized by antibody to CCR3 (CCR3 Ab). NC, negative control. Data were expressed as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant human CCL28 and recombinant human VEGFA (10 ng/ml, R&D Systems, USA) were added into culture medium.

Techniques: Saline, Ab Array, Positive Control, Western Blot, Negative Control

Journal: Scientific Reports

Article Title: Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells

doi: 10.1038/srep27152

Figure Lengend Snippet: Facing the stress of hypoxia, tumor cells up-regulate the expressions of CCL8 and VEGF, both of which could modulate functions of vascular endothelial cells through directly activating their receptors, CCR3 and VEGFR, on the cells. There are three common pathways between CCL28/CCR3 and VEGFA-VEGFR2 signaling (on the levels of PI3K-Akt, p38 MAPK and PLC gamma). As a result, CCL28 could promote angiogenesis in lung adenocarcinoma and bypass the effects of VEGFA.

Article Snippet: Recombinant human CCL28 and recombinant human VEGFA (10 ng/ml, R&D Systems, USA) were added into culture medium.

Techniques: