Journal: Oncoimmunology

Article Title: Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype

doi: 10.1080/2162402X.2015.1067744

Figure Lengend Snippet: ACEis and AGTR1 antagonist induce hypersegmentation of human neutrophils and enhance neutrophil cytotoxicity against tumor cells. (A)–(C) Neutrophils were stimulated with different concentrations of captopril. (A) Mean lobe counts of neutrophils stimulated with captopril. n = 10 donors; ** p < 0.01 compared to zero values in each time point; *** p < 0.001 compared to zero values in each time point. (B) Percentage of hypersegmented neutrophils after stimulation with captopril. n = 10 donors; ** p < 0.01 compared to zero values in each time point; ***p < 0.001 compared to zero values in each time point. (C) Representative images of Wright-Geimsa staining of neutrophils stimulated with captopril for 4 h. Hypersegmented neutrophils are denoted by arrows. (D) Top, RT-PCR analysis of ACE expression in human neutrophils: representative pictures from three healthy volunteers. Bottom, Ang II concentration in conditioned medium from neutrophils treated with either vehicle or 500 μM captopril. n = 5 donors; ***p < 0.001. (E) Mean lobe counts of neutrophils exposed to 500 μM captopril in the presence or absence of 100 nM AngII. n = 3 donors; ***p < 0.001. (F) Mean lobe counts of neutrophils subjected to ACE silencing. Neutrophils were treated with control siRNA (siCtrl) or siRNA specific for ACE (siACE) for 24 h and were further stimulated with 500 μM captopril for 4 h. Vehicle (Veh) denotes neutrophils without any stimulation. n = 3 donors; ***p < 0.001. (G) Percentage of tumor cells that survived after exposure to neutrophils. Calcein-AM-stained tumor cells were exposed to neutrophils at a ratio of 1:10 for 8 h. n = 3 donors; *p < 0.05; ***p < 0.001. (H), (I) The effect of Ang II inhibition on neutrophil hypersegmentation. Neutrophils were stimulated with either losartan, enalapril or fosinopril for 4 h. n = 3 donors; ***p < 00.001 versus vehicle. All results are shown as means ± SEMs.

Article Snippet: For evaluation of neutrophil function, neutrophils were isolated by negative selection using either neutrophil isolation kit (Miltenyi Biotec) or EasySep TM mouse neutrophil enrichment kit (StemCell technologies, Vancouver, Canada).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Concentration Assay, Control, Inhibition

Journal: Oncoimmunology

Article Title: Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype

doi: 10.1080/2162402X.2015.1067744

Figure Lengend Snippet: Neutrophil depletion reverses the captopril-induced tumor growth inhibition. (A)–(C) Effect of neutrophil depletion on tumor growth in captopril-treated mice. Starting 10 d after injection of 4T1 cells, tumor-bearing mice were administrated with 50 mg/kg/d captopril (PostCap50). Simultaneously, mice were injected with either 100 μg of anti-Ly6G antibody 1A8 (anti-Ly6G) or control IgG antibody (ctrl IgG) i.p. every 3 d. n = 10–15 for each group (A) Growth curve for tumors. *p < 0.05 for PostCap50 + ctrl IgG compared to controls; #p < 0.05 for PostCap50 + anti-Ly6G compared to PostCap50 + ctrl IgG. (B), (C) Weight of tumor and spleen. ***p < 0.001. (D) Effect of captopril on serum cytokine concentrations. Sea from naive, control, and captopril-treated mice were collected and TGF-β1, IL-2, IL-4, IFNγ and TNF-α concentrations were measured using ELISA. n = 10; n.s., not significant; *p < 0.05; **p < 0.01. All results are shown as means ± SEMs.

Article Snippet: For evaluation of neutrophil function, neutrophils were isolated by negative selection using either neutrophil isolation kit (Miltenyi Biotec) or EasySep TM mouse neutrophil enrichment kit (StemCell technologies, Vancouver, Canada).

Techniques: Inhibition, Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Oncoimmunology

Article Title: Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype

doi: 10.1080/2162402X.2015.1067744

Figure Lengend Snippet: Captopril induces neutrophil hypersegmentation in a murine tumor model. (A) Left panel, representative images of Wright-Geimsa staining of Ly6G+ cells. Right panel, mean lobe counts of Ly6G+ cells. Non, Ly6G+ cells from naive mice; Con, Ly6G+ cells from control tumor-bearing mice; Cap, Ly6G+ cells from captopril-treated tumor-bearing mice. (B)–(C) Function of hypersegmented Ly6G+ cells. Ly6G+ cells from tumor-bearing control mice or captopril-treated tumor-bearing mice were isolated and exposed to PMA for 4 h. (B) PMA-induced ROS generation from Ly6G+ cells. (C) PMA-induced NETs formation. (D) Cytotoxicity of Ly6G+ cells against 4T1 cells. Ly6G+ cells from control tumor-bearing mice or captopril-treated tumor-bearing mice were isolated and exposed to 4T1 cells at a ratio of 10:1 for 18 h. (E) Effects of captopril on Ly6G+ cells from naive mice. Blood, bone marrow, and splenic Ly6G+ cells were isolated from naive mice and exposed to 500 μM captopril for 4 h, and then mean lobe counts were measured. Veh, vehicle-treatment. (F) Cytotoxicity of captopril-treated neutrophils against 4T1 cells. Blood, bone marrow, and splenic Ly6G+ cells were isolated from naive mice and exposed to 4T1 cells at a ratio of 10:1 for 18 h in the presence or absence of 500 μM captopril. n = 5–10 mice for each group. n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001. All results are shown as means ± SEMs.

Article Snippet: For evaluation of neutrophil function, neutrophils were isolated by negative selection using either neutrophil isolation kit (Miltenyi Biotec) or EasySep TM mouse neutrophil enrichment kit (StemCell technologies, Vancouver, Canada).

Techniques: Staining, Control, Isolation

Journal: Oncoimmunology

Article Title: Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype

doi: 10.1080/2162402X.2015.1067744

Figure Lengend Snippet: Phenotype characterization of captopril-induced polarized neutrophils. (A) Basic phenotypic characteristics of mouse neutrophils. + indicates expression and - indicates lack of expression. (B) Phenotypic characterization of neutrophils. Left, the representative flow cytometry histogram for each phenotypic marker. Right, quantification of each phenotypic marker's expression. Gray, isotype-matched control IgG staining; Black, Ly6Ghigh CD11b+ cells from non-tumor-bearing mice; Blue, Ly6Ghigh CD11b+ cells from tumor-bearing mice; Red, Ly6Ghigh CD11b+ cells from captopril-treated tumor-bearing mice. n = 5–10 mice per each group; *p < 0.05; **p < 0.01; ***p < 0.001. All results are shown as means ± SEMs.

Article Snippet: For evaluation of neutrophil function, neutrophils were isolated by negative selection using either neutrophil isolation kit (Miltenyi Biotec) or EasySep TM mouse neutrophil enrichment kit (StemCell technologies, Vancouver, Canada).

Techniques: Expressing, Flow Cytometry, Marker, Control, Staining

Journal: Oncoimmunology

Article Title: Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype

doi: 10.1080/2162402X.2015.1067744

Figure Lengend Snippet: The transfer of splenic neutrophils from captopril-treated donor mice attenuates the tumor growth in recipient mice. (A) Procedure used to inject splenic neutrophils from donor tumor-bearing mice to recipient tumor-bearing mice. Donor mice harboring tumors were treated with either captopril or vehicle for 10 d. Donor splenic neutrophils were delivered to recipient tumor-bearing mice i.t. 10, 13, and 16 d after tumors were inoculated in recipient mice. (B) Growth curve for tumors in recipient mice. Recipient tumor-bearing mice were injected with Ly6G+ cells from control donor or captopril-treated donors 10, 13, and 16 d after tumor inoculation (Arrow). Control denotes tumor growth in mice injected with vehicle (RPMI). n = 5–10 mice for each group; *p < 0.05 for +Control donor versus. Vehicle; #p < 00.05 for +Captopril-treated donor versus +Control donor. (C)–(D) Tumor and spleen weights in recipient mice. n = 5–10 mice for each group; ***p < 0.001. All results are shown as means ± SEMs.

Article Snippet: For evaluation of neutrophil function, neutrophils were isolated by negative selection using either neutrophil isolation kit (Miltenyi Biotec) or EasySep TM mouse neutrophil enrichment kit (StemCell technologies, Vancouver, Canada).

Techniques: Injection, Control

Journal: Oncoimmunology

Article Title: Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype

doi: 10.1080/2162402X.2015.1067744

Figure Lengend Snippet: The mTOR pathway is involved in neutrophil hypersegmentation. (A) Inhibitory effect of rapamycin on captopril-induced hypersegmentation. Neutrophils were exposed to 500 μM captopril in the presence of PD90859 (10 μM), SB203580 (10 μM), wortmannin (1 μM), and rapamcyin (1 μM). n = 4 for each group; ***p < 0.001 compared to vehicle-treated cells; ###p < 0.001 compared to captopril-treated cells. Results are shown as means ± SEMs. (B) Western blot analysis of phosphorylation of 4E-BP1 and S6K in captopril-treated neutrophils. Neutrophils were treated with 500 μM captopril for 4 h in the presence or absence of 1 μg/mL rapamycin.

Article Snippet: For evaluation of neutrophil function, neutrophils were isolated by negative selection using either neutrophil isolation kit (Miltenyi Biotec) or EasySep TM mouse neutrophil enrichment kit (StemCell technologies, Vancouver, Canada).

Techniques: Western Blot, Phospho-proteomics