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Fig. 5
Anti-interleukin-8 (αIL-8) decreased CCL5 and anti-vascular endothelial growth factor (αVEGF) increased interleukin-8 (IL-8) and CCL2, which affected the dissemination of estrogen receptor negative (ER−) breast cancer cells (BCC). For monolayer co-cultures, breast pre-adipocytes were differentiated for 5 days before MDA-MB-231 cells were added at 3 × 103 cells/well. For zebrafish experiments, breast pre-adipocytes were differentiated for 12 days and MDA-MB-231 cells were labeled with 4 µg/ml Fast DiI™ oil red dye before injected into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) MDA-MB-231 cells were co-cultured with 50% breast adipocytes (BAd) in the presence or absence of αIL-8, αVEGF, or control isotype (Iso) antibodies at 1 µg/ml during 3 days, and secreted cytokines were quantified as described in Section “Materials and Methods,” n = 4 in each group. (B) MDA-MB-231 cells were injected in zebrafish embryos alone or in combination with 50% BAd ± anti-CCL2 (αCCL2), anti-CCL5 (αCCL5), or control Iso antibodies at 0.1 mg/ml, as described in Section “Materials and Methods.” BCC dissemination was analyzed 3 days post-injections, n = 20–24 in each group. Representative images of zebrafish embryos are shown. Arrows show disseminated BCC. BV = blood vessels. Results are presented as mean ± SEM, Student’s t-test, *p < 0.05, **p < 0.01. Data are representative of at least two independent experiments.