Journal: Cancer Growth and Metastasis

Article Title: Neutrophil-Derived Interleukin 16 in Premetastatic Lungs Promotes Breast Tumor Cell Seeding

doi: 10.1177/1179064417738513

Figure Lengend Snippet: Neutrophils are recruited in premetastatic lungs corresponding to day 7 after the subcutaneous injection of 4T1 cells. (A) Schematic representation of the xenograft model protocol. Balb/C mice were subcutaneously injected with 4T1 cells (tumor-bearing) or medium alone (Ctrl). (B) Representative Xenogen IVIS and histologic analyses of lungs of mice sacrificed at days 3, 7, 9, 14, and 21 after subcutaneous injections of 4T1 tumor cells. Scale bar represents 2,5 mm. (C) Representative Xenogen IVIS analysis of lung primary cultures obtained from tumor-bearing mice sacrificed on days 7 and 9 following the primary tumor implantation. (D) Analysis of cross-linked collagen stained with picro-red staining in tumor-bearing and corresponding control lungs. Results are expressed in mean ± SEM, * P < .05, Student t test (n = 5). (E) LOX messenger RNA expression in tumor-bearing (n = 5) and control lungs evaluated by reverse transcription-polymerase chain reaction. Results are expressed in mean ± SEM, * P < .05; Student t test (n = 5). (F) Analysis of gelatinase production by zymography. Results are expressed as mean ± SEM, * P < .05, ** P < .01, *** P < .001; Student t test. (G) Neutrophil counts in bronchoalveolar lavage (BAL) presented as the percentage within 300 cells in BAL of mice. Results are expressed as mean ± SEM. * P < .05, ** P < .01, *** P < .001; Student t test (n = 5).

Article Snippet: Murine 4T1 mammary tumor cells expressing luciferase (clone 1A1; Xenogen Corporation, Almeda, CA, USA) and murine endothelial SVEC4.10 cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% l -glutamine.

Techniques: Injection, Tumor Implantation, Staining, Control, RNA Expression, Reverse Transcription, Polymerase Chain Reaction, Zymography

Journal: Cancer Growth and Metastasis

Article Title: Neutrophil-Derived Interleukin 16 in Premetastatic Lungs Promotes Breast Tumor Cell Seeding

doi: 10.1177/1179064417738513

Figure Lengend Snippet: Neutrophils expressed IL-16 in premetastatic lungs. (A) Representative immunofluorescence experiments showing a colocalization between neutrophil foci and IL-16–positive area in premetastatic lungs. Scale bar corresponds to 100 µm. (B) Representative flow cytometry plots showing the purity of neutrophil isolation by MACS technologies. (C) Representative cytocentrifugation of neutrophils obtained using MACS technologies. Scale bar represents 100 µm. (D) IL-16 dosage by enzyme-linked immunosorbent assay in culture supernatant of neutrophils treated or not with 4T1-conditioned medium. Results are expressed as mean ± SEM. ** P < .01, Student t test. (E) Neutrophil percentage in bronchoalveolar lavage of tumor-bearing mice treated with a Ly6G blocking antibody or a control isotype. Results are expressed as mean ± SEM. ** P < .01, Mann-Whitney test (n = 6). (F) IL-16 dosage in lungs of tumor-bearing mice treated with a Ly6G blocking antibody or a control isotype. Results are expressed as mean ± SEM. * P < .05, Student t test (n = 6). IL-16 indicates interleukin 16; MACS, magnetic-activated cell sorting.

Article Snippet: Murine 4T1 mammary tumor cells expressing luciferase (clone 1A1; Xenogen Corporation, Almeda, CA, USA) and murine endothelial SVEC4.10 cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% l -glutamine.

Techniques: Immunofluorescence, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, MANN-WHITNEY, FACS

Journal: Cancer Growth and Metastasis

Article Title: Neutrophil-Derived Interleukin 16 in Premetastatic Lungs Promotes Breast Tumor Cell Seeding

doi: 10.1177/1179064417738513

Figure Lengend Snippet: Soluble factors derived from 4T1 cells induced IL-16 production in the pulmonary microenvironment. (A) ELISA against IL-16 on whole lung protein homogenates obtained from mice intratracheally instilled with control medium or 4T1-conditioned medium. Results are expressed as mean ± SEM. * P < .05, Mann-Whitney test (n = 6). (B) Percentage of cell content in BAL of mice intratracheally instilled with control medium or 4T1-conditioned medium (EC: epithelial cells, NEU: neutrophils, EO: eosinophils, LT: lymphocytes, and MP: macrophages). Results are expressed in mean ± SEM. * P < .05, Student t test (n = 6). (C) Representative Xenogen IVIS analysis and bioluminescence quantification of lungs obtained from mice treated with control medium or 4T1-conditioned medium and receiving an intravenous injection of 4T1 cell. Results are expressed as mean ± SEM. * P < .05, Student t test (n = 6). IL-16 indicates interleukin 16.

Article Snippet: Murine 4T1 mammary tumor cells expressing luciferase (clone 1A1; Xenogen Corporation, Almeda, CA, USA) and murine endothelial SVEC4.10 cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% l -glutamine.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY, Injection

Journal: Cancer Growth and Metastasis

Article Title: Neutrophil-Derived Interleukin 16 in Premetastatic Lungs Promotes Breast Tumor Cell Seeding

doi: 10.1177/1179064417738513

Figure Lengend Snippet: IL-16 improved the adhesion, migration and invasion of 4T1 cells. (A) Protocol for adhesion assay. (B) Impact of increasing concentrations of IL-16 on 4T1 cell adhesion on a confluent murine endothelial SVEC4.10 cell monolayer. Results are expressed as mean ± SEM. ** P < .01, Kruskal-Wallis test. (C) Representative pictures corresponding to each condition tested in the adhesion assay. Scale bar corresponds to 200 µm. (D) Representative pictures obtained by scratch assay. Scale bar corresponds to 200 µm. (E) Quantification of the effect of increasing concentrations of IL-16 on 4T1 cell migration evaluated by scratch assay. Ctrl+ condition corresponds to a 4T1 migration in presence of complete supplemented medium. Wound closure was expressed in percentage regarding the scratch area reported at the beginning of the experiment. Results are expressed in mean ± SEM. # P < .05 (vehicle vs IL-16, 10 ng/mL), $ P < .05 (vehicle vs IL-16, 20 ng/mL), *** P < .001 (vehicle vs Ctrl+), 1-way ANOVA. (F) Representative pictures obtained for the invasion assay. Scale bar corresponds to 100 µm. (G) Quantification of the effect of increasing concentrations of IL-16 on 4T1 invasion across a Matrigel-coated insert. * P < .05, 1-way ANOVA. (H) Expression of CD9 at the 4T1 cell surface evaluated by flux cytometry. ANOVA indicates analysis of variance; IL-16, interleukin 16.

Article Snippet: Murine 4T1 mammary tumor cells expressing luciferase (clone 1A1; Xenogen Corporation, Almeda, CA, USA) and murine endothelial SVEC4.10 cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% l -glutamine.

Techniques: Migration, Cell Adhesion Assay, Wound Healing Assay, Invasion Assay, Expressing, Cytometry