αccl2 antibody  (Bio X Cell)

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a, KEGG pathway analysis of RNA-seq data showing enriched pathways in shEzh2 compared to shRen KPC1 orthotopic PDAC tumor cells FACS sorted from C57BL/6 female mice treated with trametinib (1mg/kg) and palbociclib (100mg/kg) for 2 weeks (n=5-6 per group). b, Heatmap of RNA-seq analysis of SASP gene expression in tumor cells FACS sorted from KPC1 orthotopic PDAC tumors harboring indicated shRNAs and treated as in (a) (n=5-6 per group). c, qRT-PCR analysis of Ccl2 expression in KPC1 PDAC cells engineered to overexpress (O/E) a Ccl2 cDNA or Empty control vector (n=3 per group). A.U., arbitrary units. d, NK cell migration assay in the presence of conditioned media from KPC1 PDAC cells engineered to overexpress Ccl2 or Empty vector and treated with vehicle or trametinib (25nM) and palbociclib (500nM) for 8 days (n=3 per group). e, Flow cytometry analysis of NK cell numbers in KPC1 orthotopic PDAC tumors expressing control Empty or Ccl2 vectors following treatment as in (b) (e, Empty V, n=4; Empty TP, n=9; CCL2 O/E, n=11 and CCL2 O/E/TP, n=12 independent mice). Data represents pool of 3 independent experiments. f, Kaplan-Meier survival curve of mice with KPC1 orthotopic PDAC tumors expressing control Empty (left) or Ccl2 (right) vectors treated with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or an NK1.1 depleting antibody (PK136; 250 μg) (f, Empty V, n=12; Empty TP, n=14; Empty TP+αNK1.1, n=13; CCL2O/E V, n=10; CCL2 O/E TP, n=11 and CCL2 O/E TP+αNK1.1, n=12 independent mice). Data represents pool of 2 independent experiments. g, Flow cytometry analysis of NK cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CCL2 depleting antibody (2H5; 200 μg) for 2 weeks (g, V, n=3; αCCL2, n=3; TP, n=7 and TP αCCL2, n=9 independent mice). Data represents pool of 2 independent experiments. h, Waterfall plot of the response of shEzh2 KPC1 orthotopic PDAC tumors to treatment as in (g) (h, shEzh2 TP, n=22 and shEzh2 TP αCCL2, n=23 independent mice). Data represents pool of 3 independent experiments. i, Kaplan-Meier survival curve of mice with shEzh2 KPC1 orthotopic PDAC tumors treated as in (g) (i, shEzh2 V, n=13; shEzh2 TP, n=15 and shEzh2 TP αCCL2, n=10 independent mice) Values for shEzh2 V and T/P treated cohorts are the same displayed in Figs. 5h and ​and5i.5i. Dotted line indicates when mice were taken off of treatment. Data represents pool of 2 independent experiments. j, Flow cytometry analysis of CD4+ and CD8+ T cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CXCR3 depleting antibody (CXCR3-173; 200 μg) for 2 weeks (j, V, n=8; αCXCR3, n=5; TP, n=8; and TP+αCXCR3, n=12 independent mice). Data represents pool of 2 independent experiments. k, Waterfall plot of the response of shEzh2 KPC1 orthotopic PDAC tumors to treatment as in (j) (k, V, n=11; αCXCR3, n=7; TP, n=13; and TP+αCXCR3, n=15 independent mice). Data represents pool of 2 independent experiments. P values in a were calculated using two-sided, hypergeometric test, c,d,e,g,h,j,k using two-tailed, unpaired Student’s t-test, and f and i using log-rank test. Error bars, mean ± SEM.

Article Snippet: For neutralization of chemokine signaling, mice were injected IP with an αCCL2 (200 μg; 2H5, BioXcell) or αCXCR3 (200 μg; CXCR3-173, BioXcell) antibody twice per week.

Techniques: RNA Sequencing, Gene Expression, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation, Cell Migration Assay, Flow Cytometry, Two Tailed Test

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a, IHC staining of KPC1 orthotopic PDAC tumors harboring shRen or shEzh2 shRNAs treated with vehicle or combined trametinib (1mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Quantification of blood vessels per field are shown on inset (a, shRen V, n=4; shRen TP, n=4; shEzh2 V, n=2; shEzh2 TP, n=4 independent tumors). Scale bar, 50μm. b-c, Flow cytometry analysis of NK cell activation markers (b) and CD4+ and CD8+ T cell numbers (c) in KPC1 orthotopic PDAC tumors expressing control Empty or Ccl2 vectors and treated as in (a) (b-c, Empty V, n=4; Empty TP, n=9; Empty CCL2O/E V, n=11; CCL2 O/E TP, n=12 independent mice). Data represents pool of 3 independent experiments d, Flow cytometry analysis of CD4+ and CD8+ T cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CCL2 depleting antibody (2H5; 200 μg) for 2 weeks (d, shEzh2 V, n=3; shEzh2 αCCL2, n=3; shEzh2 TP, n=7; shEzh2 TP+αCCL2, n=9 independent mice). Data represents pool of 2 independent experiments. e, Flow cytometry analysis of NK cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CXCR3 depleting antibody (CXCR3-173; 200 μg) for 2 weeks (d, V, n=8; TP, n=8; αCCXR3, n=5; TP+αCCXR3, n=12 independent mice). Data represents pool of 2 independent experiments. P values in a-e were calculated using two-tailed, unpaired Student’s t-test. Error bars, mean ± SEM.

Article Snippet: For neutralization of chemokine signaling, mice were injected IP with an αCCL2 (200 μg; 2H5, BioXcell) or αCXCR3 (200 μg; CXCR3-173, BioXcell) antibody twice per week.

Techniques: Immunohistochemistry, Flow Cytometry, Activation Assay, Expressing, Control, Two Tailed Test

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a, KEGG pathway analysis of RNA-seq data showing enriched pathways in shEzh2 compared to shRen KPC1 orthotopic PDAC tumor cells FACS sorted from C57BL/6 female mice treated with trametinib (1mg/kg) and palbociclib (100mg/kg) for 2 weeks (n=5-6 per group). b, Heatmap of RNA-seq analysis of SASP gene expression in tumor cells FACS sorted from KPC1 orthotopic PDAC tumors harboring indicated shRNAs and treated as in (a) (n=5-6 per group). c, qRT-PCR analysis of Ccl2 expression in KPC1 PDAC cells engineered to overexpress (O/E) a Ccl2 cDNA or Empty control vector (n=3 per group). A.U., arbitrary units. d, NK cell migration assay in the presence of conditioned media from KPC1 PDAC cells engineered to overexpress Ccl2 or Empty vector and treated with vehicle or trametinib (25nM) and palbociclib (500nM) for 8 days (n=3 per group). e, Flow cytometry analysis of NK cell numbers in KPC1 orthotopic PDAC tumors expressing control Empty or Ccl2 vectors following treatment as in (b) (e, Empty V, n=4; Empty TP, n=9; CCL2 O/E, n=11 and CCL2 O/E/TP, n=12 independent mice). Data represents pool of 3 independent experiments. f, Kaplan-Meier survival curve of mice with KPC1 orthotopic PDAC tumors expressing control Empty (left) or Ccl2 (right) vectors treated with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or an NK1.1 depleting antibody (PK136; 250 μg) (f, Empty V, n=12; Empty TP, n=14; Empty TP+αNK1.1, n=13; CCL2O/E V, n=10; CCL2 O/E TP, n=11 and CCL2 O/E TP+αNK1.1, n=12 independent mice). Data represents pool of 2 independent experiments. g, Flow cytometry analysis of NK cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CCL2 depleting antibody (2H5; 200 μg) for 2 weeks (g, V, n=3; αCCL2, n=3; TP, n=7 and TP αCCL2, n=9 independent mice). Data represents pool of 2 independent experiments. h, Waterfall plot of the response of shEzh2 KPC1 orthotopic PDAC tumors to treatment as in (g) (h, shEzh2 TP, n=22 and shEzh2 TP αCCL2, n=23 independent mice). Data represents pool of 3 independent experiments. i, Kaplan-Meier survival curve of mice with shEzh2 KPC1 orthotopic PDAC tumors treated as in (g) (i, shEzh2 V, n=13; shEzh2 TP, n=15 and shEzh2 TP αCCL2, n=10 independent mice) Values for shEzh2 V and T/P treated cohorts are the same displayed in Figs. 5h and ​and5i.5i. Dotted line indicates when mice were taken off of treatment. Data represents pool of 2 independent experiments. j, Flow cytometry analysis of CD4+ and CD8+ T cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CXCR3 depleting antibody (CXCR3-173; 200 μg) for 2 weeks (j, V, n=8; αCXCR3, n=5; TP, n=8; and TP+αCXCR3, n=12 independent mice). Data represents pool of 2 independent experiments. k, Waterfall plot of the response of shEzh2 KPC1 orthotopic PDAC tumors to treatment as in (j) (k, V, n=11; αCXCR3, n=7; TP, n=13; and TP+αCXCR3, n=15 independent mice). Data represents pool of 2 independent experiments. P values in a were calculated using two-sided, hypergeometric test, c,d,e,g,h,j,k using two-tailed, unpaired Student’s t-test, and f and i using log-rank test. Error bars, mean ± SEM.

Article Snippet: For neutralization of chemokine signaling, mice were injected IP with an αCCL2 (200 μg; 2H5, BioXcell) or αCXCR3 (200 μg; CXCR3-173, BioXcell) antibody twice per week.

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation, Cell Migration Assay, Flow Cytometry, Two Tailed Test

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a, IHC staining of KPC1 orthotopic PDAC tumors harboring shRen or shEzh2 shRNAs treated with vehicle or combined trametinib (1mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Quantification of blood vessels per field are shown on inset (a, shRen V, n=4; shRen TP, n=4; shEzh2 V, n=2; shEzh2 TP, n=4 independent tumors). Scale bar, 50μm. b-c, Flow cytometry analysis of NK cell activation markers (b) and CD4+ and CD8+ T cell numbers (c) in KPC1 orthotopic PDAC tumors expressing control Empty or Ccl2 vectors and treated as in (a) (b-c, Empty V, n=4; Empty TP, n=9; Empty CCL2O/E V, n=11; CCL2 O/E TP, n=12 independent mice). Data represents pool of 3 independent experiments d, Flow cytometry analysis of CD4+ and CD8+ T cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CCL2 depleting antibody (2H5; 200 μg) for 2 weeks (d, shEzh2 V, n=3; shEzh2 αCCL2, n=3; shEzh2 TP, n=7; shEzh2 TP+αCCL2, n=9 independent mice). Data represents pool of 2 independent experiments. e, Flow cytometry analysis of NK cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or a CXCR3 depleting antibody (CXCR3-173; 200 μg) for 2 weeks (d, V, n=8; TP, n=8; αCCXR3, n=5; TP+αCCXR3, n=12 independent mice). Data represents pool of 2 independent experiments. P values in a-e were calculated using two-tailed, unpaired Student’s t-test. Error bars, mean ± SEM.

Article Snippet: For neutralization of chemokine signaling, mice were injected IP with an αCCL2 (200 μg; 2H5, BioXcell) or αCXCR3 (200 μg; CXCR3-173, BioXcell) antibody twice per week.

Techniques: Immunohistochemistry, Flow Cytometry, Activation Assay, Expressing, Two Tailed Test

Journal: Gut

Article Title: Cross-talk between the gut microbiota and monocyte-like macrophages mediates an inflammatory response to promote colitis-associated tumourigenesis

doi: 10.1136/gutjnl-2020-320777

Figure Lengend Snippet: Colonic MLM accumulation is controlled by CCL2/CCR2. (A) Expression of chemokine receptors in each macrophage subset in mice of 9 weeks after AOM-DSS induction (n=4–9). *p<0.05; ***p<0.001; ****p<0.0001, two-way ANOVA. (B) Normalised gene expression for chemokine and chemokine receptor in the colon throughout disease progression (n=6 for each time point). Data were represented as the ratio to baseline. Two-way ANOVA. (C, D) Effect of CCR2 inhibitor (C) or αCCL2 (D) on MLM accumulation and colon tumourigenesis (scale bar, 5 mm; n=6–9 per group). *p<0.05; **p<0.01, Student’s t-test. AOM-DSS, azoxymethane-dextran sodium sulfate; ANOVA, analysis of variance; CCR2, C-C chemokine receptor type 2; MLM, monocyte-like macrophage.

Article Snippet: In select experiments, mice were treated with a neutralising αCCL2 mAb (10 mg/kg/every 3 days, i.p., clone 2H5; Bioxcell) or C-C chemokine receptor type 2 (CCR2) inhibitor (propagermanium, 10 mg/kg/day, dissolved in drinking water; Sigma-Aldrich).

Techniques: Expressing, Gene Expression, Biomarker Discovery

Journal: Gut

Article Title: Cross-talk between the gut microbiota and monocyte-like macrophages mediates an inflammatory response to promote colitis-associated tumourigenesis

doi: 10.1136/gutjnl-2020-320777

Figure Lengend Snippet: TLR4 activation mediates CCL2 upregulation in CECs, MLM accumulation and tumour formation. (A) Representative images of tumours in the colon of mice treated with H 2 O or ABX (scale bar, 5 mm). The right panel indicates quantification of tumours in mice (AOM-DSS wk9: n=6; AOM-DSS +ABX wk9: n=10). ****p<0.0001, Student’s t-test. (B) CCL2 expression in colon tissues (AOM-DSS wk9: n=8; AOM-DSS +ABX wk9: n=10). *p<0.05, Student’s t-test. (C) CD206 expression in colonic macrophages (AOM-DSS wk9: n=14; AOM-DSS +ABX wk9: n=10). ****p<0.0001, Student’s t-test. (D) Colonic MLMs in mice treated with H 2 O or ABX (AOM-DSS wk9: n=6; AOM-DSS +ABX wk9: n=8). **p<0.01, Student’s t-test. (E, F) Normalised gene expression of inflammation-related genes in colon tissues (E), and Ki-67 expression in CECs (F) from vehicle or ABX-treated AOM-DSS wk9 mice (n=5–8 per group). *p<0.05; ***p<0.001, Student’s t-test. (G) Intracellular CCL2 levels in different cell types as assessed by flow cytometry (AOM-DSS wk9: n=6; AOM-DSS +ABX wk9: n=6). ****p<0.0001, two-way ANOVA. (H) CCL2 expression in CECs isolated from mice treated with H 2 O or ABX (AOM-DSS wk9: n=9; AOM-DSS +ABX wk9: n=7). *p<0.05, Student’s t-test. (I) Immunofluorescence staining of CCL2 and EpCAM in colon sections from mice treated with H 2 O or ABX. (J) PRR-related gene expression in H 2 O- or ABX-treated mice (AOM-DSS wk9: n=7; AOM-DSS +ABX wk9: n=7). **p<0.01, ****p<0.0001, two-way ANOVA. (K) Isolated CECs from normal mice were cultured in the presence of faecal extracts, or a combination of TLR4 inhibitor. CCL2 production was measured by flow cytometry (n=10 for each group). ****p<0.0001, one-way ANOVA. (L) Faecal LPS levels (AOM-DSS wk9: n=12; AOM-DSS +ABX wk9: n=6). **p<0.01, Student’s t-test. (M) ABX-pretreated AOM-DSS mice were additionally administered with rCCL2 or TLR4 ligand or vehicle. Colonic MLMs in mice (9 weeks after AOM-DSS induction) were quantified by flow cytometry (n=8–11 for each group). *p<0.05, **p<0.01, one-way ANOVA. (N–O) ABX-treated AOM-DSS mice receiving rCCL2 or TLR4 ligand. In some experiments, TLR4 ligand-treated mice also received αCCL2 or CCR2 inhibitor. Representative images (N) and quantification (O) of tumours in different treatments (scale bar, 5 mm; n=8–11 for each group, 9 weeks after AOM-DSS induction). *p<0.05, one-way ANOVA. ABX, antibiotic; AOM-DSS, azoxymethane-dextran sodium sulfate; ANOVA, analysis of variance; CCR2, C-C chemokine receptor type 2; CECs, colonic epithelial cells; IL1β, interleukin 1β; LPS, lipopolysaccharide; MLM, monocyte-like macrophage; TLR4, Toll-like receptor 4; TNF-α, tumour necrosis factor α.

Article Snippet: In select experiments, mice were treated with a neutralising αCCL2 mAb (10 mg/kg/every 3 days, i.p., clone 2H5; Bioxcell) or C-C chemokine receptor type 2 (CCR2) inhibitor (propagermanium, 10 mg/kg/day, dissolved in drinking water; Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Gene Expression, Flow Cytometry, Isolation, Immunofluorescence, Staining, Cell Culture

Journal: Gut

Article Title: Cross-talk between the gut microbiota and monocyte-like macrophages mediates an inflammatory response to promote colitis-associated tumourigenesis

doi: 10.1136/gutjnl-2020-320777

Figure Lengend Snippet: Diverse effects of stage-specific microbiota on MLM accumulation and tumourigenesis. (A) The proportion of gram-negative bacteria within microbiomes of stools from mice at different time points, as predicted by BugBase (n=4 for each time point). (B) Mice pretreated with ABX for 4 weeks were (i) repopulated with faeces from healthy baseline mice, (ii) repopulated with faeces from 3 week, 6 week or 9 week AOM-DSS mice. Mice were then induced with AOM-DSS. TLR4 and CCL2 levels in CECs, and colonic MLM levels were determined by flow cytometry (n=7–11 for each group). (C, D) The ABX- or vehicle-pretreated mice were repopulated with faeces from different stages of tumourigenesis, or sham-repopulated. Mice were subsequently induced with AOM-DSS. Representative images (C) and quantitative analysis (D) of tumours in different treatments (scale bar, 5 mm; n=7–12 for each group). Data are represented as mean±SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA. (E, F) Species differences in patients with ulcerative colitis (UC) (E) or colorectal cancer (CRC) (F) compared with healthy donors. Species name in red: known gram-negative bacteria. Runs indicate objects containing sequencing run data files, and each dot in plots represents one related run. ‘Related runs’ refer to the total number of runs that are detected positive for specific bacterial species. Multiple t-test with two-stage step-up method of Benjamini, Krieger and Yekutieli. ABX, antibiotic; AOM-DSS, azoxymethane-dextran sodium sulfate; ANOVA, analysis of variance; CECs, colonic epithelial cells; MLM, monocyte-like macrophage.

Article Snippet: In select experiments, mice were treated with a neutralising αCCL2 mAb (10 mg/kg/every 3 days, i.p., clone 2H5; Bioxcell) or C-C chemokine receptor type 2 (CCR2) inhibitor (propagermanium, 10 mg/kg/day, dissolved in drinking water; Sigma-Aldrich).

Techniques: Bacteria, Flow Cytometry, Sequencing

Journal: Gut

Article Title: Cross-talk between the gut microbiota and monocyte-like macrophages mediates an inflammatory response to promote colitis-associated tumourigenesis

doi: 10.1136/gutjnl-2020-320777

Figure Lengend Snippet: TLR4 ligands derived from microbiota induce the accumulation and activation of MLMs. (A) Functional profiling of microbiome throughout disease progression (n=4 for each time point). (B) Faecal LPS levels (AOM-DSS baseline/wk3/wk6/wk9: n=8/7/6/12). (C) CCL2 expression in CECs isolated from normal mice treated with multiple concentrations of LPS for 6 hours. control: n=10; n=3 for each group of LPS treatment. (D) Intracellular CCL2 levels in CECs from AOM-DSS mice treated with LPS, or a combination of TLR4 inhibitor (n=5–6 for each group, 9 weeks after AOM-DSS induction). (E) Mice were treated with 2.5% DSS for 7 days. Colonic MLMs were sorted by flow cytometry on day seven and then cultured in the presence of LPS, or a combination of TLR4/COX-2 inhibitor for 36 hours. MACS-sorted splenic CD4 + cells from normal mice were cultured with supernatant from MLM-drug cocultures under Th17 polarising conditions. As indicated, antimouse IL-1β was additionally added (n=8–15 for each group). (F–H) AOM-DSS mice with LPS or vehicle treatment. In some experiments, LPS (2 mg/kg)-treated mice were additionally administered with a TLR4 or COX-2 inhibitor. (F) Pro-IL1β + MLMs, IL-17A + CD4 + cells and Ki-67 + CECs in mice (9 weeks after AOM-DSS induction) were determined by flow cytometry (n=6–8 for each group). Representative images (G) and quantitative analysis (H) of tumours (scale bar, 5 mm; n=4–20 for each group). Data are represented as mean±SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA. A/D, AOM-DSS. ABX, antibiotic; AOM-DSS, azoxymethane-dextran sodium sulfate; ANOVA, analysis of variance; CCR2, C-C chemokine receptor type 2; CECs, colonic epithelial cells; IL1β, interleukin 1β; LPS, lipopolysaccharide; MLM, monocyte-like macrophage; Th17, interleukin-17-producing T-helper; TLR4, Toll-like receptor 4.

Article Snippet: In select experiments, mice were treated with a neutralising αCCL2 mAb (10 mg/kg/every 3 days, i.p., clone 2H5; Bioxcell) or C-C chemokine receptor type 2 (CCR2) inhibitor (propagermanium, 10 mg/kg/day, dissolved in drinking water; Sigma-Aldrich).

Techniques: Derivative Assay, Activation Assay, Functional Assay, Biomarker Discovery, Expressing, Isolation, Control, Flow Cytometry, Cell Culture