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

Abstract

The premetastatic niche in distant organs prior to metastatic cell arrival emerged as an important step in the metastatic cascade. However, molecular mechanisms underlying this process are still poorly understood. In particular, whether neutrophil recruitment at a premetastatic stage promotes or inhibits metastatic cell seeding has to be clarified. We aimed at unraveling how neutrophil infiltration in lung parenchyma induced by the distant primary tumor influences the establishment of lung metastasis. Elevated neutrophil counts and IL-16 levels were found in premetastatic lungs in a syngenic mouse model using 4T1 tumor cells. 4T1 cell-derived soluble factors stimulated IL-16 secretion by neutrophils. The functional contribution of IL-16 is supported by metastasis burden reduction in lungs observed on instillation of an IL-16 neutralizing antibody. Moreover, IL-16 promotes in vitro 4T1 cell adhesiveness, invasiveness, and migration. In conclusion, at a premetastatic stage, neutrophil-derived IL-16 favors tumor cell engraftment in lung parenchyma.

Keywords: IL-16; Tumor microenvironment; breast cancer; lungs; metastasis; neutrophils.

Figure 1.

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).

Figure 2.

IL-16 level is increased in lungs at a premetastatic stage. (A) Chemokine array performed on pooled total protein extracts obtained from premetastatic and control lungs (n = 5) and dot quantification (right panel). (B) IL-16 measurement by ELISA performed on lungs protein extracts. (C) Representative Western blot of secreted IL-16 production at day 7 in lungs of tumor-bearing or control mice (blot performed on pooled total protein extracts obtained, n = 9-10). Actin serves as a loading control. ELISA anti-IL-16 performed on (D) serum samples and (E) bronchoalveolar lavage fluids obtained from tumor-bearing mice or control mice at day 7. Results are expressed as mean ± SEM. **P < .01, Student t test (n = 10). (F) Representative immunohistochemistry against IL-16 performed on lung sections obtained from tumor-bearing and control mice sacrificed at days 7 and 21 following the primary tumor implantation. Scale bar represents 200 µm. ELISA indicates enzyme-linked immunosorbent assay; IL-16, interleukin 16.

Figure 3.

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.

Figure 4.

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.

Figure 5.

Impact of the IL-16 depletion on the pulmonary metastasis occurrence. (A) Schematic representation of the protocol for the IL-16 blocking antibody administration to tumor-bearing mice. (B) Comparison of the primary tumor weight (left panel) and volume (right panel) after administration of the IL-16 blocking antibody or a control isotype. Student t test (n=6). (C) Representative Xenogen IVIS analysis and bioluminescence quantification of lungs obtained from tumor-bearing mice treated with an IL-16 blocking antibody or a control isotype. Results are expressed in mean ± SEM. *P < .05, Mann-Whitney test (n = 6). (D) Representative histologic sections and tumor density quantification in lungs obtained from tumor-bearing mice treated with an IL-16 blocking antibody or a control isotype. Scale bar represents 2 mm. Results are expressed in mean ± SEM. *P < .05, Mann-Whitney test (n = 6). IL-16 indicates interleukin 16.

Figure 6.

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.