Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Immunohistochemical preparations of adjacent sections of a D-12 tumour. An avidin-biotin peroxidase-based method was used for staining. Haematoxylin was used for counterstaining. IL-8 positive foci visualized by anti-IL-8 antibody staining ( A ) and hypoxic foci visualized by anti-pimonidazole antibody staining ( B ).

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques: Immunohistochemical staining, Avidin-Biotin Assay, Staining

Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Hypoxia, IL-8 expression and neovascularization in metastatic and non-metastatic D-12 primary tumours. Points represent single tumours. Density of hypoxic foci ( A ), density of IL-8 positive foci ( B ) and density of vascular hot spots ( C ).

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Effects of anti-VEGF treatment, anti-IL-8 treatment, and combined anti-VEGF and anti-IL-8 treatment on development of hypoxia, neovascularization and spontaneous pulmonary metastasis in D-12 tumours. ( A ) Percentage of mice with metastasis. Points represent single experiments involving 10 mice each. ( B ) Densities of hypoxic foci and vascular hot spots. Columns represent mean values of 20 mice. Bars represent s.e.m.

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques:

Journal: Molecular human reproduction

Article Title: Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro.

doi: 10.1093/molehr/7.9.859

Figure Lengend Snippet: Figure 1. Localization of interleukin-8 (IL-8) in endometriotic lesions by immunohistochemistry. Sections were incubated with polyclonal rabbit anti-IL-8 antibody, then with biotin-conjugated anti-rabbit antibody and streptavidin–peroxidase. The immunoreaction was revealed with DAB as chromogen, and haematoxylin which stains the nucleus in blue was used for counterstaining. Note the intense brown positive immunostaining in endometriotic glands and stroma from (A) an ovarian endometriotic lesion, stage IV, proliferative phase, day 4 (patient no. 6, Table I), and (B) the inner lining of ovarian endometrioma, stage IV, secretory phase, day 22 (patient no. 10, Table I). (C and D) control sections incubated with normal rabbit IgG instead of the primary polyclonal rabbit antibody showing no immunostaining. Scale bar 50 µm.

Article Snippet: Immunostaining was performed using a polyclonal rabbit anti-IL-8 antibody [10 μg/ml in PBS containing 1% bovine serum albumin (BSA)] (Biosource International, Camarillo, CA, USA), a biotin-conjugated affinity-purified goat anti-rabbit antibody (H L) (2 μg/ml) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), peroxidase-conjugated streptavidin (1.3 μg/ml) (Jackson ImmunoResearch Laboratories), diaminobenzidine (Sigma Chemical Co, St Louis, MO, USA) as chromogen and haematoxylin for counterstaining.

Techniques: Immunohistochemistry, Incubation, Immunostaining, Control

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Combination therapy with anti-programmed cell death 1 antibody plus angiokinase inhibitor exerts synergistic antitumor effect against malignant mesothelioma via tumor microenvironment modulation.

doi: 10.1016/j.lungcan.2023.107219

Figure Lengend Snippet: Fig. 1. MPM cells secreted multiple angiogenic factors. A, Comprehensive analysis of the expression of transcripts of multiple angiogenic factors (VEGFA, PDGFb, FGF2, HGF, ANG1 and IL-8) in human umbilical vein endothelial cell (HUVEC), mesothelial cell (Met-5A) and MPM cell lines (MSTO-211H, H2452, H2052, and H28) by qRT-PCR. Expression of FGF2 transcripts in all MPM cells was higher than in HUVECs. B, Comprehensive analysis of the protein expression of angiogenic factor receptors (VEGFR2, PDGFRβ, FGFR1, c-Met, Tie2 and CXCR1) in HUVEC, human mesothelial cell and MPM cells by immunoblotting. MPM cells expressed higher level of FGFR1 compared with HUVECs. C, Antiproliferative effect of multi-angiokinase inhibitor nintedanib (NTD) in MPM cells. Mesothelial cells and MPM cells were treated with serially diluted NTD for up to 72 h. The relative numbers of viable cells were quantified using CCK-8 assay. Points, mean % viable cells; bars, SEM of at least three independent experiments performed in triplicate.

Article Snippet: Rabbit polyclonal Ab against CXC motif chemokine receptor 1 (CXCR1) (#55450–1- AP) was obtained from Proteintech (Rosemont, IL).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay

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: Extracellular in vivo levels of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) were significantly increased in human breast cancer. A total of 17 breast cancer patients underwent microdialysis before surgery. One catheter was inserted into the breast cancer, and another catheter was inserted into adjacent normal breast tissue as control. Proteins were analyzed by Luminex in cohort 1 (expressed as pg/ml), n = 6 and by proximity extension assay (PEA) technology in cohort 2 (expressed as linear NPX), n = 11 as described in Section “ .” The bars to the right represent data from both cohorts normalized to the control in each cohort (relative protein expression), n = 17. (A) IL-8 in vivo measurements in microdialysis samples from breast cancer patients. (B) VEGF in vivo measurements in microdialysis samples from breast cancer patients. Results are presented as mean ± SEM and were analyzed pairwise by Wilcoxon signed-rank test, * p < 0.05, *** p < 0.001. IL-8 and VEGF exhibited a significant positive correlation by Spearman’s correlation test, r = 0.51 and p < 0.05.

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: In Vivo, Luminex, Expressing

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

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) 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 × 10 6 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 “ ” ( 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 “ ,” 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 “ .” 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 “ .” 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 “ .” 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 “ ,” n = 17–21 in each group. (G) Number of co-disseminated T47D/neutrophil cells was quantified as described in Section “ ,” n = 21–26 in each group. (H) Number of co-disseminated MDA-MB-231/neutrophil cells was quantified as described in Section “ ,” 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.

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: Expressing, Immunocytochemistry, Cell Culture, Incubation, Labeling, Injection, Staining

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: 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 “ ,” 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 “ ,” 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.

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: Expressing, Cell Culture, Labeling, Injection, Recombinant, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, In Vitro, Negative Control