Figure 3.

Growth of EO771 cells in syngeneic mice following treatment with a Cxcr₂ antagonist. (a) Syngeneic 57BL/6 mice were inoculated in the mammary gland with 1 × 10⁶ at five weeks of age, and injected i.p. daily with vehicle (blue) or 2 mg/kg (red) or 20 mg/kg (green) SB225002 beginning 11 days after cell inoculation. SB225002 completely suppressed tumor growth after 14 days. Differences between vehicle- and 2 mg/kg SB225002-treated mice were not significantly different (P = 0.145); differences between vehicle- and 20 mg/kg SB225002-treated mice were significantly different (P = 0.005) by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N = 5 per group. (b) Immune gene expression in tumors 17 days after treatment with 20 mg/kg SB225002. Shown is the relative expression in control and SB225002-treated mice in comparison to their changes in EO771/shPlac1 cells (Table 1). (c) FACS analysis of immune cell tumor infiltrates in isografts after treatment with vehicle or SB225002 as in (b). SB225002 treatment reduced the percentage of immune cell tumor infiltrates of CD11b⁺/Gr-1⁺ myeloid-derived suppressor cells (MDSC) and Foxp3⁺/CD25⁺ T cells (Treg), and increased the percentages of CD8⁺/CD4⁺ T cells (T), CD3⁺/NK1.1⁺ NK cells (NK) and F4/80⁺/CD80/86⁺ macrophages (Mϕ) and CD11c⁺/CD80/86⁺ dendritic cells (DC). Numbers in parentheses ( ) represent the percentages of each cell population. (d) Bar graph represents the mean±SD of the percent distribution of immune cell tumor infiltrates as in (c); P values were determined by the unpaired two-tailed Student’s t test, N = 4 per group. (e) CD8⁺ T cell infiltration determined by IHC in tumor isografts from vehicle-treated (EO771/Ctl) and SB225002-treated (EO771/SB) mice. Infiltration of CD8⁺ T cells increased after treatment with 20 mg/kg SB225002. Magnification 600X. (f) Macrophage (F4/80) and Treg cell (Foxp3) infiltration, Plac1 expression and apoptosis by cleaved caspase-3 expression (Caspase) in tumor isografts from vehicle-treated (EO771/Ctl) and SB225002-treated (EO771/SB) mice. Infiltration of macrophages and Treg cells were reduced and apoptosis was increased after treatment with 20 mg/kg SB225002. Magnification 400X