Fig. 6

Anti-interleukin-8 (αIL-8) reduced lymphocyte function-associated antigen 1 (LFA-1) expression in neutrophils and the neutrophil-mediated dissemination of breast cancer cells (BCC) in the presence of breast adipocytes (BAd). For immunocytochemistry, neutrophils were cultured at 1 × 10⁶ cells/ml in BAd-conditioned or control medium and incubated 45 min at 37°C. Prior zebrafish injections, breast pre-adipocytes were differentiated for 12 days, BCC were labeled with 4 µg/ml Fast DiI™ oil red dye, and neutrophils were labeled with 6 µg/ml DiB. All BCC were injected ± αIL-8, anti-LFA-1 (αLFA-1), or isotype (Iso) control antibodies at 0.1 mg/ml into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) Neutrophils were cultured ± conditioned medium (CM) from BAd ± αIL-8 or Iso control at 1 µg/ml, and whole cells were stained with anti-human LFA-1 as described in Section “Materials and Methods” (n = 15 random fields per group). Insets show magnification of the cells. (B) Breast pre-adipocytes were differentiated during 12 days and cultured in DMEM supplemented medium during 24 h, and secreted interleukin-8 (IL-8) was measured in control and CM as described in Section “Materials and Methods,” n = 4 in the CM group. (C) MCF-7 cells were injected in zebrafish embryos together with 33% BAd ± 33% neutrophils, as described in Section “Materials and Methods.” BCC dissemination was analyzed at 1 day post-injections, n = 11–25 in each group. (D) T47D cells were injected in zebrafish embryos together with 33% BAd ± 33% neutrophils, as described in Section “Materials and Methods.” BCC dissemination was analyzed at 1 day post-injections, n = 15–26 in each group. (E) MDA-MB-231 cells were injected in zebrafish embryos together with 33% BAd ± 33% neutrophils, as described in Section “Materials and Methods.” BCC dissemination was analyzed at 1 day post-injections, n = 12–21 in each group. (F) Number of co-disseminated MCF-7/neutrophil cells was quantified as described in Section “Materials and Methods,” n = 17–21 in each group. (G) Number of co-disseminated T47D/neutrophil cells was quantified as described in Section “Materials and Methods,” n = 21–26 in each group. (H) Number of co-disseminated MDA-MB-231/neutrophil cells was quantified as described in Section “Materials and Methods,” n = 12–21 in each group. Results are presented as mean ± SEM, Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are representative of at least two independent experiments.