Figure 7.

Interleukin-8 (IL-8) increased dissemination and MUC-1 expression in estrogen receptor positive (ER+) breast cancer cells (BCC) and IL-8 gene silencing affected the dissemination and expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and mucin-1 (MUC-1) integrins in estrogen receptor negative (ER−) BCC. Prior injections, MCF-7 and T47D cells were cultured 48 h in β-estradiol (E2) 1 nM. All BCC were labeled with 4 µg/ml Fast DiI™ oil red dye and then injected into the perivitelline space of 2 days old zebrafish embryos, which express enhanced green fluorescent protein in endothelial cells. (A) MCF-7 cells were injected into zebrafish embryos ± recombinant human IL-8 (rhIL-8) at 1 µg/ml. BCC dissemination was analyzed after 3 days post-injections as described in Section “Materials and Methods,” n = 18–20 in each group. (B) Western blot analysis of MCF-7 cells treated ± rhIL-8 at 10 ng/ml during 5 days to evaluate the expression of MUC-1, VCAM-1, and ICAM-1. GAPDH load control was reused for illustrative purposes. (C) T47D cells were injected into zebrafish embryos ± rhIL-8 at 1 µg/ml. BCC dissemination was analyzed after 3 days post-injections as described in Section “Materials and Methods,” n = 17–23 in each group. (D) Western blot analysis of T47D cells treated ± rhIL-8 at 10 ng/ml during 3 days to evaluate the expression of MUC-1, VCAM-1, and ICAM-1. GAPDH is shown as load control. (E) MDA-MB-231 cells transfected with or without an IL-8 gene silencer RNA were injected into the perivitelline space of zebrafish embryos, and BCC dissemination was analyzed at 3 days post-injections, n = 19–22 in each group. (F) Western blot analysis of MDA-MB-231 cells transfected with/without an IL-8 silencer RNA (siIL-8) to evaluate the expression of MUC-1, VCAM-1, and ICAM-1. GAPDH load control was reused for illustrative purposes. (G) ELISA quantification of extracellular in vitro IL-8 in cell culture supernatants of MDA-MB-231 cells transfected with negative control silencer RNA (siRNA Control) or IL-8 silencer RNA (siRNA IL-8) during 2 days showed the successful knockdown of IL-8, n = 6 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 < 0.0001. Western blots shown in this figure were prepared by cropping and pasting from original membranes shown in full in Supplementary Figure 1. Data are representative of at least two independent experiments.