anti human neutralizing il 6 antibody  (Proteintech)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2 regulates the phenotypic plasticity of CAFs and promote PDAC progression by IL6. A Gene Set Enrichment Analysis (GSEA) of affected signatures in fibroblasts (circCUL2 vs. control). B GSEA plots for inflammatory CAF (iCAF) signatures in circCUL2 overexpression NFs from control. C - D qRT–PCR analysis of iCAF markers (IL6, TNF-α and IL1α) and myCAF marker (Acta2 and Axin2) expression in circCUL2 overexpression NFs. E Flow cytometric analysis of PDGFRα and α-SMA expression in circCUL2 overexpression NFs. F - H . Representative cytokine arrays for circCUL2-overexpression NFs and control (n = 3). arrows indicate the cytokines with significant changes, which were further confirmed by qRT–PCR and ELISA. I - L EdU assay ( I ), colony formation ( J ), Scratch wound healing assays ( K ) and transwell assays ( L ) of PANC-1 cells treated with conditioned medium circCUL2-overexpression NFs or anti-IL6. Scale bar, 100 μm. M western blot analysis of STAT3 and p-STAT3 in PANC-1 cells. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Over Expression, Quantitative RT-PCR, Marker, Expressing, Enzyme-linked Immunosorbent Assay, EdU Assay, Western Blot, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2-overexpression NFs promote PDAC progression in vivo. A Representative Bioluminescence images, lung and HE staining of lung tissue of mice 4 weeks after tail vein injection of luc-PANC-1 cell treated with conditioned medium as indicated (n = 8 per group). Scale bar, 100 μm. B Relative luminescence intensity in each group. C Histogram analysis of the metastatic nodules number in per lung. D lung metastasis rate of each group (Chi-square test). E - F Representative bioluminescence images and histogram analysis of luminescence intensity in each at day 30 are shown (n = 6). G Abdominal metastasis rate was calculated for indicated group (Chi-square test). H Representative images of orthotopic model in each group on which autopsy was performed. Red arrow indicated primary tumor; S, spleen; T, primary tumor; M, metastasis. I Images of PDX from 2 patients in 5 mice. ( J ) Tumor growth curves of indicated group (n = 5). K qRT–PCR analysis of circCUL2 levels in PDX of mice before and after treatment. L Representative images of IHC for PDGFRα and IL6. Scale bar, 100 μm. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Over Expression, In Vivo, Staining, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: MyD88 is a direct downstream target gene of miR-203a-3p. A - B Colony formation and transwell assays of PANC-1 cells treated with conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. Scale bar: 100 μm. C - D ELISA assays detected IL6 level of conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. E Venn analysis of the potential downstream target genes of miR-203a-3p, predicted by miRTarbase, miRWalk and Tarbase. F - G qRT–PCR analysis of screened downstream target genes of miR-203a-3p in indicated NFs and CAFs. H Luciferase reporter assay was used to detect the luciferase activity of MyD88-Wild Type (MyD88-wt) and miR-203a-3p binding site mutated MyD88 (mYD88-mut) luciferase reporter cotransfected with miR-203a-3p mimic. N.S., no significant. I - J Western blotting analysis of MyD88, p65, pp65, IKBα and p-IKBα expression after overexpression of circCUL2 or silence of miR-203a-3p in NFs, or silence of circCUL2 or overexpression of miR-203a-3p in CAFs. GAPDH as a loading control. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2 promotes proliferation and migration via MyD88/NF-κB/IL6 axis. A - D Cotransfection of circCUL2 overexpression plasmid and miR-203a-3p mimic in NFs or circCUL2 siRNA and miR-203-3p inhibitor in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα, and secretion level of IL6. E - F PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with miR-203a-3p mimic, or CAFs cotransfected circCUL2 siRNA and miR-203-3p inhibitor for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. G - J Cotransfection of circCUL2 overexpression plasmid and MyD88 siRNA in NFs or circCUL2 siRNA and MyD88 overexpression plasmid in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα and secretion level of IL6. K - L PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with MyD88 siRNA, or CAFs cotransfected circCUL2 siRNA and MyD88 overexpression plasmid for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Migration, Cotransfection, Over Expression, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: Clinical implication of circCUL2/miR-203a-3p/IL6 axis in PDAC. A , F , K The relative expression of miR-203a-3p ( A ), MyD88 ( F ) and IL6 ( G ) in 161 PDAC tissues compared to paired NAT. B - C , G - H , L - M Association analysis between miR-203a-3p ( B - C ), MyD88 ( G - H ) and IL6 ( L - M ) expression levels and LN status and tumor stages in 161 PDAC tissues. ( D - E , I - J , N - O ) Kaplan-Meier analysis of the correlation between miR-203a-3p ( D - E ), MyD88 ( I - J ) and IL6 ( N - O ) expression levels and OS or DFS of 161 PDAC patients. The median miR-203a-3p, MyD88 and IL6 expression was used as the cutoff value. P Proposed model indicates the mechanism by which circCUL2 activated iCAF phenotype and production of IL6 to promote malignant progression of PDAC via miR-203a-3p/MyD88/NF-κB pathway. Data are expressed as the mean ± SD. * p < 0.05 and *** p < 0.001

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Expressing

Journal: Theranostics

Article Title: Tumor-neuroglia interaction promotes pancreatic cancer metastasis

doi: 10.7150/thno.42440

Figure Lengend Snippet: Schwann cells secret IL6 to promote pancreatic cell migration, invasion, and epithelial-mesenchymal transition in vitro . A . QRT-PCR analysis of inflammatory cytokine mRNAs ( TNFA , IL1A , IL1B , IL6 , IL8 , LIF , CCL2 ) in human SCs cultured in monolayers or co‑cultured with pancreatic cancer cells for 24 h. B . The expression of corresponding receptors in pancreatic cancer cells ( IL6R , GP130 , IL1R1 , CXCR1 , and CXCR2 ) cultured in monolayers or co‑cultured with human SCs was assessed by qRT-PCR. C . The protein levels of IL6 in cell supernatants were determined via an ELISA assay. D . Representative images of wound healing assay showing that the addition of IL6 neutralizing antibodies (50 ng/mL), rather than isotype control antibodies (50 ng/mL), to co-cultured CM (co-CM) impaired the co-CM-induced pancreatic cells migration. Medium supplemented with 1% FBS was used as a control. E and F . Representative images of Transwell migration and invasion assays showing that the addition of IL6 neutralizing antibodies (50 ng/mL), rather than isotype control antibodies (50 ng/mL), to co-CM abrogated the co‑CM-induced pancreatic cancer cell migration and invasion. Medium containing 1% FBS was used as a control. Scale bar: 100 μm. G . Cancer cells were cultured in control medium (3% FBS/DMEM) or 70% co-CM (co-CM:10% FBS/DMEM = 7:3) in the presence of isotype control antibodies (50 ng/mL) or 50 ng/mL IL6 neutralizing antibodies for 48 h and then collected for to determine the protein levels off E-cadherin, N-cadherin, and Snail using immunoblotting analysis. GAPDH was used as loading control. H . Representative images of IHC staining of IL6 and S100 in sequential tissues from two PDAC patients. Scale bar: 20 µm. I . Correlations between the gene expression of IL6 and EMT markers ( CDH1 , CDH2 , VIM , SNAI1 , ZEB1 , ZEB2 ) in PDAC were obtained using the publicly accessible cBioportal tool (TCGA PanCancer Atlas). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The drugs and reagents used in this study comprised: Inhibitor of NF-κ kinase subunit beta (IKKβ) inhibitor ML120B (MedChemExpress, Monmouth Junction, NJ, USA), anti-human IL6 neutralizing antibody (R&D Systems, Minneapolis, MN, USA), anti-human IL1β neutralizing antibody (Abcam, Cambridge, MA, USA), recombinant human IL1β (Peprotech, Rocky Hill, NJ, USA), and recombinant human tumor necrosis factor alpha (TNFα) (Peprotech).

Techniques: Migration, In Vitro, Quantitative RT-PCR, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Western Blot, Immunohistochemistry

Journal: Theranostics

Article Title: Tumor-neuroglia interaction promotes pancreatic cancer metastasis

doi: 10.7150/thno.42440

Figure Lengend Snippet: STAT3 signaling is critical for SC-induced pancreatic cancer cell migration and invasion. A . Immunoblotting assay demonstrating increased STAT3 phosphorylation in pancreatic cancer cells following exposure to co-cultured conditioned medium (co-CM) for the indicated times. Cells without co-CM treatment were used as a control (Ctrl). B . Western blotting of pancreatic cancer cells shows reduced level of phosphorylated STAT3 (p-STAT3) following the addition of IL6 neutralizing antibodies (50 ng/mL) to co-CM compared with those only treated with co-CM. The duration of co-CM treatment in both groups was 30 min. Cells with no treatment were used as a control. C . Representative images of the immunofluorescence assay results for anti-p-STAT3 (red) and DAPI (blue) staining. Pancreatic cancer cells were treated with co-CM or co-CM plus IL6 neutralizing antibodies (50 ng/mL) for 30 min and then collected for immunofluorescence analysis. Cells without any treatment were used as a control. Scale bar: 10 μm. D . Immunoblotting analysis showing disrupted STAT3 activation following co-CM incubation at the indicated times in STAT3 siRNA#1 targeted pancreatic cancer cells. Scrambled siRNA targeted cancer cells exposed to co‑CM were used as a control. E and F . Representative images of wound healing assay showing that STAT3 interference undermined co-CM induced pancreatic cancer cell migration. Control siRNA targeted tumor cells incubated with co-CM were used as a control. G and H . Scrambled siRNA and STAT3 siRNA- targeted MIA PaCa-2 and AsPC-1 cells were used for Transwell migration and invasion assays. Co-CM was used as a chemoattractant in all groups. Scale bar: 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The drugs and reagents used in this study comprised: Inhibitor of NF-κ kinase subunit beta (IKKβ) inhibitor ML120B (MedChemExpress, Monmouth Junction, NJ, USA), anti-human IL6 neutralizing antibody (R&D Systems, Minneapolis, MN, USA), anti-human IL1β neutralizing antibody (Abcam, Cambridge, MA, USA), recombinant human IL1β (Peprotech, Rocky Hill, NJ, USA), and recombinant human tumor necrosis factor alpha (TNFα) (Peprotech).

Techniques: Migration, Western Blot, Cell Culture, Immunofluorescence, Staining, Activation Assay, Incubation, Wound Healing Assay

Journal: Theranostics

Article Title: Tumor-neuroglia interaction promotes pancreatic cancer metastasis

doi: 10.7150/thno.42440

Figure Lengend Snippet: Active NF-κB signaling is responsible for elevated IL6 production from SCs. A . Human SCs were cultured in control medium (3% FBS/DMEM) or 70% TCM (TCM: 10% FBS/DMEM = 7:3) for 24 h and subsequently collected for qRT-PCR analysis. B . Human SCs were treated with TCM at the indicated times and then collected for immunoblotting analysis of phosphorylated p65 (p-p65) and total p65 (t-p65). Cells without TCM exposure were used as a control. Loading control: GAPDH. C . Western blotting analysis of p-p65, t-p65 and total IkBα in human SCs cultured in control medium or TCM in the absence or presence of 40 μmol/L IKKβ inhibitor (IKKβi) ML120B for 15 min. Loading control, GAPDH. D . QRT-PCR analysis of IL1B , IL6 , and IL8 mRNA expression in human SCs cultured in control medium or 70% TCM in the absence or presence of 40 μmol/L IKKβ inhibitor (IKKβi) ML120B for 24 h. E . Representative IHC images showing intensive p-NF-κB/p-65 staining in S100-positive SCs in surgically resected PDAC samples, but not in SCs in the adjacent non-cancerous tissues from two patients. Scale bar: 20 μm. F . Differential analysis of p-NF-κB/p-65 IHC score between PDAC samples (N = 49) and adjacent non-cancerous tissues (N = 25). An unpaired Students' t test was applied for statistical analysis. G . Representative IHC images showing substantial overlap of IL6 and p-NF-κB/p-65 staining in PDAC tissues from two patients. Scale bar: 20 μm. H . Correlation analysis of IL6 and p-NF-κB/p-65 IHC score in SCs (N = 49). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The drugs and reagents used in this study comprised: Inhibitor of NF-κ kinase subunit beta (IKKβ) inhibitor ML120B (MedChemExpress, Monmouth Junction, NJ, USA), anti-human IL6 neutralizing antibody (R&D Systems, Minneapolis, MN, USA), anti-human IL1β neutralizing antibody (Abcam, Cambridge, MA, USA), recombinant human IL1β (Peprotech, Rocky Hill, NJ, USA), and recombinant human tumor necrosis factor alpha (TNFα) (Peprotech).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Staining

Journal: Theranostics

Article Title: Tumor-neuroglia interaction promotes pancreatic cancer metastasis

doi: 10.7150/thno.42440

Figure Lengend Snippet: IL1β derived from pancreatic cells activates the NF-κB pathway in SCs and upregulates pro-inflammatory cytokine production from SCs. A . Immunoblotting analysis of p‑p65 and t-p65 in human SCs cultured in control medium or TCM in the absence or presence of 40 ng/mL isotype control antibodies or IL1β neutralizing antibodies for 15 min. Loading control, GAPDH. B . QRT‑PCR analysis of IL1B , IL6 , and IL8 mRNA levels in human SCs cultured in control medium or 70% TCM in the absence or presence of 40 ng/mL isotype control antibodies or IL1β neutralizing antibodies for 24 h. C . Western blotting was performed to confirm the knockdown efficiency of IL1R1 in human SCs treated with two IL1R 1-targeted siRNAs. Loading control, GAPDH. D . Immunoblotting analysis of p-p65 and t-p65 in human SCs showing that knockdown of IL1R1 in SCs impaired NF-κB (p65) pathway activation after TCM treatment. GAPDH was used as the loading control. E . QRT-PCR analysis of IL1B , IL6 , and IL8 mRNA levels in scrambled siRNA or IL1R1 -specific siRNA targeted human SCs cultivated with 70% TCM for 24 h. F . siRNA mediated IL1B knockdown in MIA PaCa-2 and AsPC-1 cells was verified using ELISA. G . Immunoblotting analysis of p-p65 and t-p65 in human SCs exposed to control medium, control TCM, or TCM derived from IL1B knockdown pancreatic cancer cells for 15 min. Loading control, GAPDH. H . QRT-PCR analysis of IL1B , IL6 , and IL8 mRNA levels in human SCs cultured in control 70% TCM or 70% TCM derived from IL1B knockdown pancreatic cancer cells for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The drugs and reagents used in this study comprised: Inhibitor of NF-κ kinase subunit beta (IKKβ) inhibitor ML120B (MedChemExpress, Monmouth Junction, NJ, USA), anti-human IL6 neutralizing antibody (R&D Systems, Minneapolis, MN, USA), anti-human IL1β neutralizing antibody (Abcam, Cambridge, MA, USA), recombinant human IL1β (Peprotech, Rocky Hill, NJ, USA), and recombinant human tumor necrosis factor alpha (TNFα) (Peprotech).

Techniques: Derivative Assay, Western Blot, Cell Culture, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Tumor-neuroglia interaction promotes pancreatic cancer metastasis

doi: 10.7150/thno.42440

Figure Lengend Snippet: The tumor-neuroglia interaction facilitates cancer dissemination in vivo . A . Schematic illustration of the animal experiment design. In brief, luc-AsPC-1 cells were cultured alone or co-cultivated with human SCs, or co-cultured with human SCs in the presence of IL6 or IL1β neutralizing antibodies. Forty-eight hours later, luc-AsPC-1 cells were digested and injected into the lateral tail vein of SCID mice. Four weeks after injection, lung metastasis was detected using an in vivo imaging system. B . Numbers of mice that developed lung metastasis in each group at the end of experiment are shown. C . Representative images of mice that developed lung metastasis in each group are shown, as visualized using the Bruker In-Vivo Xtreme imaging system. D . Quantitative analysis of the bioluminescence intensity from the lung were acquired using the Bruker molecular imaging software. E . At the end of animal experiment, all mice were sacrificed and their lungs were excised, photographed, and sectioned. The upper panel presents the macroscopic appearance of metastatic lung tumors; the lower panel presents the H&E staining. Scale bar:100 μm. F . Statistical analysis of metastatic nodules in the lung. G . Schematic illustration of the crosstalk between SCs and pancreatic cancer cells. Tumor cells secrete IL1β to activate the NF-κB pathway in SCs and increase IL6 production from SCs, which binds to IL6R on cancer cells and promotes PCa cell metastasis via the activation of STAT3 signaling. In summary, SCs could acquire a tumor-promoting phenotype by interacting with pancreatic cancer cells.

Article Snippet: The drugs and reagents used in this study comprised: Inhibitor of NF-κ kinase subunit beta (IKKβ) inhibitor ML120B (MedChemExpress, Monmouth Junction, NJ, USA), anti-human IL6 neutralizing antibody (R&D Systems, Minneapolis, MN, USA), anti-human IL1β neutralizing antibody (Abcam, Cambridge, MA, USA), recombinant human IL1β (Peprotech, Rocky Hill, NJ, USA), and recombinant human tumor necrosis factor alpha (TNFα) (Peprotech).

Techniques: In Vivo, Cell Culture, Injection, In Vivo Imaging, Imaging, Software, Staining, Activation Assay