Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Addition of breast adipocytes (BAd) significantly increased interleukin-8 (IL-8) secretion but decreased vascular endothelial growth factor (VEGF) secretion compared to breast cancer cells (BCC) cultured alone and anti-IL-8 and anti-VEGF significantly decreased BAd-induced angiogenesis in primary tumors in zebrafish. BCC were cultured in 3D spheres alone or in combination with BAd. Secreted IL-8 and VEGF were analyzed as described in Section “ .” Prior injections, breast pre-adipocytes were differentiated for 12 days and estrogen receptor positive (ER+) BCC were cultured ± β-estradiol (E2) 1 nM for 48 h. All BCC were labeled with 4 µg/ml Fast DiI™ oil red dye. Cells were injected ± anti-IL-8, anti-VEGF, or isotype control at 0.1 mg/ml ± E2 1 nM into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) BAd mammospheres alone and low metastatic ER+ MCF-7 ± 90% BAd mammospheres were cultured ± E2 1 nM during 7 days, n = 4–5 in each group. (B) BAd mammospheres alone an ER+ T47D with intrinsically higher metastatic capacity ± 90% BAd mammospheres were cultured ± E2 1 nM during 7 days, n = 5–6 in each group. (C) Estrogen receptor negative (ER−) metastatic MDA-MB-23 ± 90% BAd and BAd mammospheres were cultured during 7 days, n = 4–5 in each group. (D) MCF-7 cells were injected alone or in combination with 50% BAd ± E2 1 nM, tumor angiogenesis was analyzed 3 days post-injections, n = 12–18 in each group. (E) T47D cells were injected alone or in combination with 50% Bad ± E2 1 nM, tumor angiogenesis was analyzed 3 days post-injections, n = 10 in each group. (F) MDA-MB-231 cells were injected alone or in combination with 50% BAd, tumor angiogenesis was analyzed 3 days post-injections, n = 7–10 in each group. Representative confocal images are shown for each cell line. BV = blood vessels. Results are presented as mean ± SEM and analyzed by 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.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Cell Culture, Labeling, Injection

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Anti-interleukin-8 (IL-8) treatment significantly decreased breast cancer cells (BCC) dissemination induced by breast adipocytes (BAd). Prior injections, breast pre-adipocytes were differentiated for 12 days and estrogen receptor positive (ER+) BCC were cultured ± β-estradiol (E2) 1 nM for 48 h. All BCC were labeled with 4 µg/ml Fast DiI™ oil red dye. Cells were injected ± anti-IL-8, anti-VEGF, or isotype control at 0.1 mg/ml ± E2 1 nM into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) MCF-7 cells were injected alone or in combination with 50% BAd ± E2 1 nM. BCC dissemination was evaluated 3 days post-injections, n = 18–27 in each group. (B) T47D cells were injected alone or in combination with 50% BAd ± E2 1 nM. BCC dissemination was evaluated 3 days post-injections, n = 16–28 in each group. (C) MDA-MB-231 cells were injected alone or in combination with 50% BAd. BCC dissemination was evaluated 3 days post-injections, n = 14–18 in each group. (D) MCF-7, T47D, and MDA-MB-231 cells were cultured alone or in combination with 50% breast pre-adipocytes in vitro during 24 h, and migration of cells was determined as described in Section “ ,” n = 6 in each group. BV = blood vessels. Arrows indicate disseminated BCC. Results are presented as mean ± SEM and analyzed by Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Cell Culture, Labeling, Injection, In Vitro, Migration

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Anti-interleukin-8 (αIL-8) decreased vascular endothelial growth factor (VEGF) and CCL5 secretion, which affected estrogen receptor positive (ER+) breast cancer cells (BCC) dissemination. For monolayer co-cultures, breast pre-adipocytes were differentiated for 5 days before ER+ BCC were added at 4 × 10 3 cells/well. For zebrafish experiments, breast pre-adipocytes were differentiated for 12 days, MCF-7 cells were cultured + β-estradiol (E2) 1 nM for 48 h and 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) MCF-7 cells were co-cultured with 50% breast adipocytes (BAd) in the presence or absence of αIL-8, anti-VEGF (αVEGF), or control isotype (Iso) antibodies at 1 µg/ml during 3 days in the presence of E2 1 nM, and secreted cytokines were quantified as described in Section “ ,” n = 5–4 in each group. (B) T47D cells were co-cultured with 50% BAd in the presence or absence of αIL-8, αVEGF, or control Iso antibodies at 1 µg/ml during 3 days in the presence of E2 1 nM, and secreted cytokines were quantified as described in Section “ ,” n = 6–5 in each group. (C) MCF-7 cells were injected in zebrafish embryos alone or in combination with 50% BAd ± anti-CCL5 (αCCL5) or Iso control antibody at 0.1 mg/ml and E2 1 nM, as described in Section “ .” MCF-7 dissemination was analyzed 3 days post-injections, n = 13–27 in each group. Representative images of zebrafish embryos are shown. Arrows show disseminated BCC cells. BV = blood vessels. Results are presented as mean ± SEM, Student’s t -test, * p < 0.05, *** p < 0.001. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Cell Culture, Labeling, Injection

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: 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 × 10 3 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 “ ,” 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 “ .” 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.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Labeling, Injection, Cell Culture