14,15-EET induces the infiltration and tumor-promoting function of neutrophils to trigger the growth of minimal dormant metastases

Infiltrating neutrophils are known to promote in thedevelopment of tumor. However, it is unclear whether and how neutrophils areinvolved in triggering the growth of dormant metastases. Here we show that14,15-epoxyeicosatrienoic acid (14,15-EET) can trigger the growth of dormantmicrometastases by inducing neutrophilic infiltration and converting neutrophilfunction. 14,15-EET triggered neutrophil infiltration in metastatic lesions byactivating STAT3 and JNK pathways to induce the expression of human IL-8 andmurine CXCL15 in corresponding tumor cells. The continuous expression ofhIL-8/mCXCL15 was maintained by the sustained and enhanced activation of JNKpathway. 14,15-EET up-regulated miR-155 expression by activating STAT3 and JNKpathways. miR-155 in turn down-regulated the expression of SHIP1 and DET1, thusaugmenting the activation of JNK and c-Jun. Moreover, the function ofneutrophils was converted from tumor-suppressing to tumor-promoting by14,15-EET in vivo. By inducing the production of G-CSF/IL-6 in vivo, 14,15-EET induced the enhancement of STAT3 activation in neutrophilsto increase MMP-9 expression and decrease TRAIL expression. Neutrophil-derivedMMP-9 was required for 14,15-EET to induce angiogenesis during the growth ofdormant micrometastases. Depleting neutrophils or inhibiting hIL-8/mCXCL15up-regulation resulted in the failure of 14,15-EET to promote the developmentof micrometastases. These findings reveal a mechanism through which theinfiltration and tumor-promoting function of neutrophils could be induced totrigger the growth of dormant metastases, which might be a driving force forthe tumor recurrence based on dormant metastases.

,15-DHET, and t-AUCB were purchased from Cayman chemical. Mouse TGF-β1, human TGF-β1, mouse G-CSF and IL-6 were purchased from PeproTech (Rocky Hill, NJ). H 2 O 2 and HOCl were purchased from Sigma-Aldrich (St. Louis, MO). 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ), SB203580, PD98059, SP600125, and STAT3 inhibitor VIII were purchased from Merck4Biosciences (Calbiochem). All inhibitors were dissolved in DMSO as a stock solution and diluted with culture medium to the desired concentration without toxicity to cells. B16F0 cells, untreated or treated with T/H/H for 10 days, were labeled with CFSE. 5 × 10 5 CFSE-labeled cells were injected into mice via tail vein. Lungs were harvested from mice 5 h and 24 h after tumor cell injection. Frozen sections were prepared and analyzed by fluorescence microscopy. Fluorescent spots were counted from randomly chosen fields in the sections of each mouse.

Histology
Mice were anesthetized. Left ventricle was intubated and rapidly perfused with 20 ml of 0.9% saline via ascending aorta, and then continuously perfused with 30 ml paraformaldehyde with the concentration of 40 g/L. The lung tissues were harvested and embedded in paraffin according to standard histological procedures. Tissue sections were prepared and subjected to H&E staining or immunohistochemical analysis. For H&E staining, the sections were stained with hematoxylin and eosin. 5 metastatic foci in each mouse were randomly chosen for measuring the size, which was calculated using the formula: (length + width)/2. Immunohistochemical staining for proliferation marker Ki-67 was performed using anti-mouse Ki-67 antibody (Abcam Biotechnology) as primary antibody. HRP-conjugated anti-rabbit IgG was used as secondary antibody. Images were obtained using OLYMPUS-BX51 microscope at 10 × 10 or 40 × 10 magnification. Staining intensity of cells was evaluated under a microscope and graded (1, weak; 2, moderate; 3, strong) in a double blinded fashion (stainer-and examiner-blind). Staining intensity of tissue sections was assessed using a semi-quantitative immunohistochemical scoring system, HSCORE. The HSCORE was calculated using the following equation: HSCORE = ∑Pi(i + 1), where i is the staining intensity of cells and Pi is the percentage of the cells at each level of intensity [20].
For detecting neutrophils, anti-mouse Ly6G antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and HRP-conjugated secondary antibody were used for immunohistochemical analysis. Images were obtained using OLYMPUS-BX51 microscope at 10 × 10 and 40 × 10 magnification. The neutrophils were counted by using Image-pro-plus 6.0 software. The neutrophil density was defined as the number of neutrophils per microscopic field with metastatic lesion.

ELISA analysis
Cell-free supernatants from untreated or 14,15-EETtreated tumor cells were harvested at the indicated time points. hIL-8/mCXCL15 in the supernatants was quantified using human IL-8 and mouse CXCL15 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. To determine the effect of 14,15-EET on the production of G-CSF and IL-6 in vivo, mice were treated with 14,15-EET (30 μg/kg) by i.v. injection, once every two days. Serum levels of G-CSF and IL-6 were detected using mouse G-CSF and IL-6 ELISA kits (R&D Systems, Minneapolis, MN).

Western blot assay
Cells were treated with the indicated stimuli or isolated from mice. Western blot assay was done as described previously [49,50]. Primary antibodies and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), R&D systems (Minneapolis, CA), and Cell Signaling (Beverly, MA), respectively.

MMP-9 assay
To detect neutrophil-released MMP-9, neutrophils were incubated at the concentration of 5 × 10 6 /ml at 37 °C for 4 h in RPMI 1640 medium [12]. MMP-9 in supernatants was detected by gelatin zymography, and the relative activity of MMP-9 was calculated as described previously [49].

Immunofluorescence
The lung tissues were harvested at the indicated time points after tumor cell inoculation. Frozen tissue sections were prepared and subjected to immunofluorescence analysis as previously described [49]. For detecting neutrophils, anti-mouse Ly6G antibody (Santa Cruz Biotechnology) was used as primary antibody. The sections were further stained with Cy3-conjugated secondary antibody. For detecting microvessels, antimouse CD34 antibody (Santa Cruz Biotechnology) and Cy3-conjugated secondary antibody were used. Images were obtained using a laser scanning confocal microscope (Olympus, FV500, Japan).

Analysis of gene expression by real-time RT-PCR
Total RNA was extracted from cells with TRIzol reagent (Invitrogen) or lung tissues homogenized in TRIzol according to the manufacturer's instructions. For real-time RT-PCR assays, the cDNA sequences of all detected genes were retrieved from NCBI database. The primers were designed with the Oligo Primer Analysis 4.0 software and the sequences were blasted (http://blast.ncbi. nlm.nih.gov/Blast.cgi). 100 ng of total RNA was used for reverse transcription using Superscript II RNase H reverse transcriptase (Invitrogen) in a volume of 25 μl. Then 2 μl of cDNA was amplified with SYBR Green Universal PCR Mastermix (Bio-Rad, Richmond, CA) in duplicate. For sample analysis, the threshold was set based on the exponential phase of products, and C T value for samples was determined. The resulting data were analyzed with the comparative C T method for relative gene expression quantification against house keeping gene Gapdh(m) or GAPDH(h).
The sequences of the primers used for detecting gene expression were as follows:

Flow cytometric analysis
To analyze the effect of neutrophil depletion in vivo, mice were sacrificed one day after the second injection and the last injection of anti-Ly6G antibody. The heparinized blood was harvested for analysis. 20 μl of whole blood was incubated with PE-Cy7-anti-mouse CD11b, PE-antimouse Ly6G or PE-anti-mouse Ly6C, APC-anti-mouse Gr-1, and FITC-anti-mouse F4/80 (eBioscience) for 30 min on ice. RBCs were then lysed. The samples were centrifuged at 350 × g for 5 min, and then resuspended in 300 μl of PBS for flow cytometric analysis. CD11b + Ly6G + cells were considered as neutrophils.
When neutrophils were isolated using Percoll gradient, the isolated cells were assessed by flow cytometric analysis using PE-Cy7-anti-mouse CD11b and PE-anti-mouse Ly6G or PE-anti-mouse Ly6C antibodies (eBioscience).

Analysis of microRNA expression by real-time RT-PCR
Total RNA was extracted from cells with TRIzol reagent (Invitrogen). The relative quantity of microRNAs was determined by real-time RT-PCR. The resulting data were analyzed with the comparative C T method for relative microRNA expression quantification against house keeping gene Gapdh(m) or GAPDH(h).

Soft agar assay
Tumor cells were pretreated with 14,15-EET (100 nM) for 10 days. The cells were then suspended in 0.3% agar in DMEM (20% FBS) and plated (1 × 10 4 cells/ well in 6-well plates) on a layer of 0.6% agar in DMEM (20% FBS) in triplicate. After 21-day culture in the presence of 14,15-EET, the colonies of tumor cells were photographed under a microscope. (A) Control mice and the mice inoculated with T/H/H-B16F0 cells were untreated or treated with 14,15-EET. The mice were sacrificed on d42 after inoculation. The frozen sections of lung tissues were prepared and subjected to immunofluorescence analysis. The sections were stained for Ly6G (red) to identify neutrophils. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Insets are the high-power view of neutrophils in corresponding picture indicated by arrow. (B) Anti-Ly6G antibody was used to deplete neutrophils in vivo as described in Methods. The neutrophils (CD11b + Ly6G + ) in blood were detected by flow cytometry one day after the second injection ( anti-Ly6G(1) ) and last injection ( anti-Ly6G(2) ) of the antibody. The cells were also analyzed by staining for Ly6C. The monocytes (F4/80 + ) were also analyzed. (C) Mice inoculated with T/H/H-HepG2 or T/H/H-MCF-7 cells were untreated or treated with 14,15-EET. Anti-Ly6G antibody was used to deplete neutrophils in vivo when the mice were treated with 14,15-EET. The mice (n = 6 per group) were sacrificed on d42 after inoculation. Neutrophil density in lung tissue sections was determined after immunofluorescence staining (left). Metastatic nodules on the surface of lungs were counted (right). (D) Mice were inoculated with B16F1 cells (5 × 10 5 cells/ mouse). The mice received the i.v. injection of 14,15-EET (30 μg/kg), once every two days, form d1 to d19 after inoculation. Anti-Ly6G antibody was used to deplete neutrophils in vivo from d8 to d20 after inoculation. The mice (n = 6 per group) were sacrificed on d21 after inoculation. Metastatic nodules on the surface of lungs were counted. *p < 0.05, **p < 0.01. T/H/H-B16F0 cells (5 × 10 5 cells/mouse) or B16F1 cells (5 × 10 5 cells/mouse) were inoculated to mice by injection via tail vein. The mice (n = 6 per group) were sacrificed on d5 and d10 after inoculation. The sections of lung tissues were subjected to immunohistochemical staining for identifying the infiltration of neutrophils (left, Bar, 50 μm). Neutrophil density in lung tissue sections was determined after immunohistochemical staining (right). (B) The expression of CXCL15 in B16F1 cells is much higher than that in B16F0 cells. B16F0 cells and B16F1 cells were untreated or treated with 14,15-EET (100 nM) for 48 h. The expression of Cxcl15 gene was detected by realtime RT-PCR and ELISA. (C) 14,15-EET induces the conversion of neutrophil function in vivo. Mice were inoculated with B16F1 cells (5 × 10 5 /mouse) by injection via tail vein. The mice received the i.v. injection of 14,15-EET (30 μg/kg), once every two days, form -d10 before inoculation to d10 after inoculation. Anti-Ly6G antibody was used to deplete neutrophils in vivo from -d8 before inoculation to d7 after inoculation, thus depleting neutrophils in the early stage of metastasis. The mice (n = 6 per group) were sacrificed on d21 after inoculation. Metastatic nodules on the surface of lungs were counted. (D) Early depletion of neutrophils favors the formation of micrometastases by T/H/H-B16F0 cells. Mice were inoculated with T/H/H-B16F0 cells (5 × 10 5 /mouse). Anti-Ly6G antibody was used to deplete neutrophils in vivo from -d8 before inoculation to d7 after inoculation. The mice then received the i.v. injection of 14,15-EET (30 μg/kg), once every two days, form d22 to d40 after inoculation. The mice (n = 6 per group) were sacrificed on d42 after inoculation. Metastatic nodules on the surface of lungs were counted. (E) Increasing EET production in vivo promotes tumor cell metastasis. Mice were inoculated with B16F1 cells (5 × 10 5 cells/mouse) by injection via tail vein. The mice were treated with sEH inhibitor t-AUCB (oral gavage, 10 mg/kg/d), and/or 14,15-EEZE (i.v. injection, 30 μg/kg/2d), from -d10 before inoculation to d10 after inoculation. Anti-Ly6G antibody was used to deplete neutrophils in vivo from -d8 before inoculation to d7 after inoculation, thus depleting neutrophils in the early stage of metastasis. The mice (n = 6 per group) were sacrificed on d21 after inoculation. Metastatic nodules on the surface of lungs were counted. (F) Control mice (N) and the mice inoculated with T/H/H-B16F0 cells (T) were untreated or treated with 14,15-EET. On d42 after inoculation, neutrophils were isolated from the peritoneal cavity of mice after recruitment as described in Methods. The expression of Stat3 gene was detected by real-time RT-PCR and Western blot. The relative level of STAT3 to β-actin was calculated after densitometric analysis of Western blots. (G) Mice were untreated or treated with 5, 8,11,and 14,30 μg/kg) for 10 days, once every two days. The levels of serum G-CSF and IL-6 in naive mice and EET-treated mice were detected by ELISA.

Supplementary
(H) 14,15-EET cooperates with G-CSF/IL-6 to modulate the expression of Mmp9 and Trail genes in neutrophils. Neutrophils were isolated from bone marrow of naive mice, and stimulated with 14,15-EET (100 nM) and/or G-CSF/IL-6 (50 ng/ml of each) for 12 h. The expressions of Mmp9 and Trail genes in neutrophils were detected at mRNA level by real-time RT-PCR. *p < 0.05, **p < 0.01.