FIGURE 5.

Effects of SB225002 on recruitment of TANs in vivo. (A) SB225002 significantly reduced TANs in C666-1 tumor-bearing nude mice. Total numbers of 10,000 cells gated on CD45-positive cells were collected and CD45⁺CD11b⁺Ly6C^midLy6G^high cells were gated as TANs. Numbers illustrated indicate the percentage of the cells. (B) Flow cytometry analysis quantified TANs in tumor at the end of treatment with SB225002 combined radiotherapy. The percentage of TANs and the absolute number of TANs were shown as mean ± SD (n = 4; *p < 0.05; **p < 0.01; Student’s t-test). (C) TAN was assessed on paraffin-embedded C666-1 tumor sections by Ly6G immunohistochemical staining after SB225002 combined radiotherapy treatment. Representative immunohistochemistry images showing Ly6G-positive TANs in the xenograft tumors in five groups. Scale bar, 50 mm. Image analysis data for Ly6G-positive TANs were shown as mean ± SD (n = 3 animals each group, **p < 0.01; ***p < 0.001; Student’s t-test). (D,E) Intracellular flow cytometry detected the increased secretion of TGF-β, IL-10 and VEGF by Ly6G-positive neutrophils in C666-1 tumors (TAN) compared with the neutrophils in peripheral blood from both non-tumor bearing mice (nPBN) and C666-1 tumor-bearing mice (tPBN). Total numbers of 10,000 cells gated on CD45-positive cells were collected and the CD45⁺CD11b⁺Ly6C^midLy6G^high cells in tumor were gated. Numbers illustrated indicate the percentage of the cells (n = 3; *p < 0.05; **p < 0.01; Student’s t-test). (F) Schematic representation of the experiment sequence in air pouch experiments of recruitment of neutrophils stimulated by supernatants of irradiated tumor cells in vivo. (G) Flow cytometry analysis quantified neutrophils recruitment into the air pouch skin samples. Air pouch skin samples were collected at the indicated time point, and neutrophils were analyzed by flow cytometry. Total cell numbers per 10⁵ cells are shown. Means ± SD are depicted (n = 3; *p < 0.05; Student’s t-test).