anti human ccl22 mdc neutralizing ab  (R&D Systems)

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: L1CAM promotes the recruitment of Tregs by upregulating CCL22. (A) Volcano plot of differentially expressed genes (DEGs), on the basis of the median value of the L1CAM mRNA expression level. (B) The upregulated DEGs were used to perform GO enrichment analysis. (C) The secretion of cytokines and chemokines in the supernatants of shNC and shL1CAM-EC1 cells (up) or scramble and OE-L1CAM-KYSE450 cells (down) cultured for 48 h was assessed with a human multiplex bead-based kit. The expression of CCL22 was detected by qRT-PCR (D) and ELISA (E) between shNC and shL1CAM-EC1 cells, or scramble and OE-L1CAM-KYSE450 cells. (F) Expression of L1CAM and CCL22 mRNAs in the different groups tumor samples derived from Figure 2H . (G) IHC of tumor tissues in different groups derived from Figure 2H . Representative images are shown (200×). (H) The IHC scores of L1CAM and CCL22 in different groups. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Tumor cells, anti-CCL22 neutralizing antibodies (500 ng/mL; R&D, MAB336) or CCL22 recombinant protein (100 ng/mL, BioLegend, 584902) was added to the lower chamber.

Techniques: Expressing, Cell Culture, Multiplex Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: L1CAM promotes the recruitment of Tregs through CCL22 in vivo . (A) The correlation between FOXP3 and CCL22 in the ESCC TCGA database, n = 94. (B) The purity of CD4 + CD25 + CD127 - Tregs sorted from the peripheral blood of patients with ESCC, according to FACS. (C) The numbers of migrating Tregs obtained from ESCC patient blood samples was calculated for the shNC, shL1CAM, shL1CAM + recombinant protein CCL22 (100 ng/mL), shL1CAM + PBS, scramble, OE-L1CAM, OE-L1CAM + CCL22 antibody (500 ng/mL), and OE-L1CAM + IgG groups. (D) Flow chart of the in vivo experiments. (E) mRNA expression of FOXP3 and CCR4 in the indicated groups. (F) Percentage of CD4 + FOXP3 + Tregs and FOXP3 + CCR4 + Tregs recruited to tumor sites, as analyzed by flow cytometry. (G) Expression of FOXP3 in tumor tissues of different groups, determined by IHC (200×). (H) The expression of p-Akt, Akt, p-NF-κB, NF-κB, and CCL22 was detected by Western blot between shL1CAM and OE-L1CAM cells treated with or without p-Akt inhibitor (RF-04691502) and p-NF-κB inhibitor (QNZ). β-actin expression was used as a loading control. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Tumor cells, anti-CCL22 neutralizing antibodies (500 ng/mL; R&D, MAB336) or CCL22 recombinant protein (100 ng/mL, BioLegend, 584902) was added to the lower chamber.

Techniques: In Vivo, Recombinant, Expressing, Flow Cytometry, Western Blot

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: High expression of CCL22 predicts poor survival in patients with ESCC. (A–B) qRT-PCR analysis of CCL22 expression in paired fresh tissues from 47 patients with ESCC. GAPDH was used to normalize the data, which were analyzed with the 2 -ΔΔCt method. (C) Kaplan–Meier survival analysis of overall survival on the basis of high ( n = 24) and low ( n = 23) CCL22 expression by qRT-PCR. (D) The percentage of CD4 + FOXP3 + Tregs in tumor tissues obtained from patients with ESCC. Correlations between CD4 + FOXP3 + Tregs and L1CAM (E), CD4 + FOXP3 + Tregs, and CCL22 (F), L1CAM, and CCL22 in the tumor tissues of patients with ESCC (G) were evaluated with linear regression analysis. (H) Schematic diagram of the proposed molecular mechanism: L1CAM promotes the recruitment of Tregs into the tumor site through PI3K/Akt/NF-κB-CCL22-CCR4. Tregs produce TGF-β, which further induces L1CAM upregulation in ESCC tumor cells. ** P < 0.01.

Article Snippet: Tumor cells, anti-CCL22 neutralizing antibodies (500 ng/mL; R&D, MAB336) or CCL22 recombinant protein (100 ng/mL, BioLegend, 584902) was added to the lower chamber.

Techniques: Expressing, Quantitative RT-PCR