Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

This study aims to investigate the mechanisms underlying leptin-mediated crosstalk between tumor-associated macrophages (M2 macrophages) and breast cancer cells. THP1 human leukemic monocytes were induced to differentiate into M2 macrophages by PMA (100 nM) and IL-4 (20 ng/mL). Quantitative RT-PCR and Western blot revealed that leptin (100 nM) significantly increased the expression of leptin receptor (ObR) in the M2 macrophages (P < 0.01) and stimulated interleukin (IL)-8 expression in the M2 macrophages, mouse macrophage cells RAW264.7, and primary mouse peritoneal macrophages in a dose- and time-dependent manner. Leptin-induced IL-8 production was sensitive to the ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), and anti-ObR neutralizing antibody (4 μg/mL). Leptin (100 ng/mL) substantially increased the phosphorylation of p38 and ERK1/2. Thus, leptin may induce IL-8 production in M2 macrophages by interacting with ObR to activate the p38 and ERK signaling pathways. Scratch and transwell chamber assay showed that both recombinant IL-8 and leptin-induced M2 macrophage-derived IL-8 promoted the migration and invasion of human breast cancer cells MCF7 and MDA-MB-231 (All P < 0.01). In a nude mice xenograft model of breast cancer (n = 5 per group), injection of leptin (0.1 μg/g) dramatically increased tumor volume and mass, reduced survival, exacerbated pulmonary metastasis, and elevated IL-8 and Ki67 expression in the tumor tissue (All P < 0.05) compared with PBS injection. Depletion of mouse macrophage by Clophosome®-clodronate liposome and injection of anti-mouse IL-8 neutralizing antibodies in the xenograft tumor significantly attenuated those leptin-mediated stimulations (All P < 0.05). These findings indicate that leptin may promote tumor growth and metastasis by stimulating IL-8 production in tumor-associated macrophage.

The role of leptin in mediating the crosstalk between the tumor microenvironment and breast cancer cells remains unclear. Tumor-associated macrophages (M2 macrophages), Research Paper www.impactjournals.com/oncotarget which are the most notable hematopoietic cells in the tumor microenvironment, have been found to promote breast cancer progression and correlate with poor prognosis [19][20][21][22][23][24][25]. M2 macrophages can secret cancer-promoting cytokines, growth factors, and inflammatory factors [26,27]. Leptin receptor ObRb has been found to be expressed in macrophages [28]. Leptin induces macrophage recruitment to the tumor microenvironment by stimulating aromatase and estrogen receptor (ER) α in the macrophages [29]. In addition, leptin also stimulates macrophages to secret vascular endothelial growth factor and pro-inflammatory cytokines, including interleukin (IL)-1, TNF-α, and IL-6 [15,19]. Thus, leptin may induce the release of cancerpromoting factors from M2 macrophages to indirectly stimulate breast cancer progression. This study aims to test this hypothesis and further investigate the molecular mechanism underlying leptin-mediated cancer promoting effects of M2 macrophages on breast cancer cells.

Leptin induced ObR expression and IL-8 production in M2 macrophages
The morphology of THP1 cells was changed from round into anchorage-dependent shape after the cells differentiated into macrophages ( Figure 1A). Flow cytometry revealed that M2 macrophages, which were induced from THP1 cells by PMA (100 nM) and IL-4 (20 ng/mL), expressed lower levels of IL-12 (1.38%) and higher levels of TGF-β (70.97%) and IL-10 (19.63%) compared with THP1 cells and THP1 macrophages ( Figure  1B). The expression of the mannose receptor CD206, a M2 macrophage surface marker, was dramatically increased in M2 macrophages ( Figure 1B), suggesting that exposure to PMA and IL-4 appears to successfully induce THP1 cells to differentiate into M2 macrophages. Immunofluorescence staining showed that ObR was expressed weakly in THP1 cells, whereas highly expressed in both THP1 macrophages and M2 macrophages ( Figure  1C). Leptin (100 nM) significantly increased mRNA ( Figure 1D) and protein expression ( Figure 1E) of both the long form (ObRb) and the short form (ObRt) leptin receptor in M2 macrophages (All P < 0.01).

Leptin promoted breast cancer progression via stimulating IL-8 production in macrophage in nude mouse xenograft model
From day 15 to day 30 after leptin injection, mice in the leptin group developed the largest tumor volume (P < 0.05, Figure 6A). Compared with PBS injection, leptin injection significantly increased tumor mass on day 30 (P < 0.01, Figure 6B and 6C), worsened survival (P < 0.05, Figure 6D), exacerbated pulmonary metastasis (P < 0.01, Figure 6E), and elevated the expression of IL-8 and Ki67 in the tumor tissue (P < 0.01, Figure 6G).

DISCUSSION
The current study investigated the molecular mechanism underlying leptin-mediated macrophage-breast cancer interaction. The data in the current study suggest that leptin may up-regulate ObR to activate the P38/ MAPK and MAPK/ERK 1/2 signaling pathways, which in turn stimulate IL-8 promoter and IL-8 production in M2 macrophages, consequently promoting breast cancer progression ( Figure 8).
Binding of leptin to leptin receptors can trigger a series of downstream signaling events, including both canonical (MAPK/ERK 1/2, JAK2/STATs, PI-3K/AKT) and non-canonical signaling pathways (p38 MAPK, JNK, PKC and AMPK), in various types of cells [15]. Our previous study has shown that leptin stimulates IL-8 expression via activating the PI3K/AKT signaling pathway in breast cancer cells MCF7 and SKBR3 [33]. Here, we found that in M2 macrophages, the leptin-ObR interaction activated the p38/MAPK and MAPK/ERK 1/2 signaling pathways but not the PI3K/AKT pathway. In addition, in M2 macrophages, leptin-induced IL-8 expression was mainly at transcriptional level because leptin activated IL-8 promoter rapidly. Our results cannot rule out the possibility that leptin may also increase IL-8 production at translational and/or post-translational levels in M2 macrophages. Thus, leptin-ObR interaction appears to link to distinct intracellular signaling cascades in different types of cells. In the current study, to minimize the direct stimulatory effects of leptin on breast cancer cells [33], we washed the leptin-treated M2 macrophages extensively before loading them in the transwell co-culture system to analyze breast cancer cell migration and invasion. Nevertheless, residual leptin, which may remain on the M2 macrophages even after extensive wash, could diffuse into the chamber of breast cancer cells and directly induce cancer cell migration and invasion. However, our experiments in vivo showed that depleting mouse macrophages and neutralizing mouse IL-8 significantly reduced leptin-induced xenograft growth and metastasis, which clearly support that macrophage-derived IL-8 can contribute to leptin-induced breast cancer progression.
IL-8, a pro-inflammatory factor, has been shown to promote cancer progression not only by directly stimulating cancer cell proliferation, survival, and migration but also by increasing angiogenesis [31,32]. Our current study support that at least one of the sources of IL-8 appears to be the tumor microenvironment. Notably, our IHC staining of human specimens suggest that the proportions of cells with positive staining for the leptin receptor ObR, the macrophage marker CD68, and IL-8 increase as breast cancer progresses from carcinoma  showing leptin-induced ObR-dependent phosphorylation of ERK 1/2 and p38 and production of IL-8. M2 macrophages were pretreated with a polyclonal anti-human ObR antibody (4 μg/mL) for 16 hours and then treated with PBS or leptin (100 ng/mL). (E) Luciferase reporter assay to measure leptin-mediated IL-8 promoter activation. M2 macrophages were pretreated with ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative luciferase units (RLU) normalized to β-galactosidase activity are shown. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01.  In summary, our study suggest that leptin may promote breast cancer progression by stimulating IL-8 production in M2 macrophage. The leptin-ObR-IL-8 axis certainly adds another layer of complexity in the tumor microenvironmentbreast cancer interaction, which drives cancer progression. www.impactjournals.com/oncotarget

Ethics statement
The protocol for laboratory animal handling and experiments was in strict accordance with Chongqing Management Approach of Laboratory Animal (Chongqing government order NO.195). The protocol has been approved by the Institutional Review Board of Chongqing Medical University (Reference Number: CQMU 2010-26). Surgery on animals was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering.

Animals
Female nude BALB/c mice (6-8-week old) were purchased from the Center of Laboratory Animals of Chongqing Medical University (Chongqing, China). All the mice were monitored daily by animal care staff and laboratory personnel. The mice had free access to food and water and were properly treated if distress, infection, or inflammation occurred. When aggregate tumor burden exceeded 1 cm in diameter or body condition score was < 2, the mice were euthanized by CO 2 narcosis. We used a regulated flow valve (in addition to cylinder regulator) to control the CO 2 flow rate to 20% of chamber volume per minute. These methods were chosen because they caused minimal stress to the animals. The euthanasia procedure was consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

M2 macrophage induction
To induce macrophage differentiation (THP1 macrophages), THP1 cells (1 × 10 6 cells per well) were cultured in 6-well plates and treated with PMA (100 nM) for 72 hours. To induce M2 phenotype, THP1 cells were treated with PMA (100 nM) for 36 hours and then with PMA (100 nM) and IL-4 (20 ng/mL) for additional 36 hours. The morphology of these cells was observed; phase contrast images of the cells were collected under an inverted microscope.

Flow cytometry
THP1 cells were washed and re-suspended in PBS after PMA or PMA + IL-4 induction. To determine the expression of CD206, the cells were then incubated with PE-labeled anti-CD206 antibody (BioLegend, San Diego, CA, USA). To determine the intracellular expression of IL-10, IL-12, and TGF-β, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (BioLegend), and stained with PE-labeled anti-IL-10, anti-IL-12, anti-TGF-β antibodies (BioLegend). After final wash, the labeled cells were analyzed by flow cytometry on a FACScan flow cytometer (BD Biosciences).

Quantitative real-time PCR
Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcribed into firststrand complementary DNA (Invitrogen) according to the manufacturer's instruction. Quantitative real-time reverse transcription PCR (qRT-PCR) was performed according to the standard protocol of the SYBR Premix Ex Taq TM kit (TaKaRa, Japan). All the reactions were performed in a volume of 25 µL in triplicate. Primers for ObRb, ObRt, CCL2, CCL18, CCL17, IL-8, TNF-α, IL-6, IL-10, MMP9, TGF-β1, and β-actin were obtained from Invitrogen. The primer sequences are displayed in Table 1. The PCR reaction condition was: initial denaturation at 95°C for 30 seconds and 40 cycles of denature at 95°C for 5 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 30 seconds. Relative gene expression of the target gene to β-actin was calculated by normalizing the target gene level to β-actin gene level. The second derivative maximum method was used to determine the Crossing point (Cp) for each sample (in triplicate). The mean value was used to calculate the ΔCp (Cp of the target gene -Cp of β-actin gene). Expression levels of genes were obtained according to the equation: 2 -ΔΔCp .

Western blot analysis
M2 macrophages were lysed in RIPA lysis buffer (Beyotime) and the protein concentration in the cell lysate was determined by BCA method. A total of 80 µg of M2 macrophage cell lysate was separated by SDS-PAGE. The proteins were then transferred to a PVDF membrane, and the membrane was incubated with the primary antibodies against human ObR (Santa Cruz), IL-8 (Abcam), pSTAT-Tyr 705 , pAKT-Ser 473 , pERK-Thr 202 /Tyr 204 , STAT3, AKT, ERK (Cell Signaling Technology, USA), and β-actin (Santa Cruz). HRP anti-mouse or rabbit IgG (Beyotime, China) was used as the secondary antibodies. The antigen-antibody reaction was visualized by enhanced chemiluminescence (ECL) assay (VIAGENE, USA).

Migration assay (scratch assay)
Breast cancer cell migration was evaluated by the transwell co-culture system (porous polycarbonate membrane filters with 0.4 µm pore, Millipore, USA). MCF7 and MDA-MB-231 cells were cultured to confluence in the lower chamber of the 6-well plates, and a sterile pipette tip was used to scratch across the cell layer. M2 macrophages were induced with leptin (100 ng/mL, Peprotech) for 12 hours and then the M2 macrophages were washed with DMEM to remove leptin. After wash, the M2 macrophages (1 × 10 6 cells/mL per chamber) were added into the upper chamber. The M2 macrophages and breast cancer cells were co-cultured for 48 hours. Phase contrast images of the breast cancer cells were collected under a microscope.

Invasion assay
Breast cancer cell invasion was evaluated by a 24well transwell co-culture system (Millipore, USA) with 8 μm-pore polycarbonate filter membrane coated with 3.5 mg/mL matrigel (Sigma, USA). Breast cancer cells (2 × 10 4 cells/mL per well) were cultured in the upper chamber. M2 macrophages were treated with leptin (100 ng/mL) for 12 hours, collected, and washed. The M2 macrophages (2 × 10 6 cells/mL per well) were then added in the lower chamber. Breast cancer cells and M2 macrophages were co-cultured for 24 hours. Breast cancer cells inside the chamber were removed by cotton swabs and the cells passing through the membrane of the upper chamber were stained with 0.1% crystal violet for 10 minutes and counted under a microscope. A total of 5 to 10 observation views were randomly selected on each membrane and the average number of cells was calculated.

Immunohistochemistry of human breast tissue specimens
Tissue sections of human breast tissue specimens were incubated with the antibodies against human ObR, CD68, and IL-8 (Beyotime, China). Cells that showed positive staining for ObR, CD68, and IL-8 were counted. A minimum of 10 observation views at 400× magnification were randomly selected from each tissue section. The average percentage of cells with positive staining was calculated.

Luciferase reporter assay
Plasmid pGL3-basic was a generous gift from Dr. Qin Zhou (Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine, Ministry of Education Chongqing Medical University, Chongqing, China). A total of 1 × 10 5 M2 macrophages were seeded in 24-well plates and co-transfected with a total of 800 ng of plasmid pGL3-basic-IL8 and the control plasmid pGL3-basic using Lipofectamine 2000 (Invitrogen). After 48 hours of culture, the cells were lysed and the lysates were collected. Renilla and firefly luciferase activities in the cell lysate were measured with a Dual-Luciferase Reporter System (E1910, Promega, WI, USA).

Isolation of mouse peritoneal macrophages
Mice were injected intraperitoneally with 1 mL 3% thioglycollate broth (Sigma-Aldrich) to induce an inflammatory response. Three days later, the mice were injected intraperitoneally with 5 mL cold PBS to harvest macrophages. Based on our preliminary experiments, this protocol can yield a larger number of inflammatory macrophages. Approximately 1 × 10 7 macrophages can be collected from one mouse. Macrophages were isolated from peritoneal lavage by plastic adherence.

Tumor xenograft
MDA-MB-231 cells (1 × 10 7 cells for each mouse) were inoculated into the mammary fat pad of female nude mice (6-week old, n = 5 per group). When the diameter of the xenograft tumor reached 0.5 cm, mice were randomized into PBS and leptin groups. Mice in the PBS group received intratumor injection of PBS; mice in the leptin group received leptin injection in the tumor (0.1 μg/g) once every 5 day for 4 consecutive weeks. To induce macrophage depletion, mice were injected intraperitoneally with Clophosome ® -clodronate liposome (neutral) or control neutral liposome at an initial dose of 0.2 mL per mouse and then 0.1 mL per mouse once a week for 8 weeks before leptin or PBS injection. Body weight and tumor volume were measured every 5 days. Tumor volume was calculated according to the following equation: V = (L ×W 2 ) × 0.5, where "L" and "W" represent the length and width of xenografts, respectively. Mice were sacrificed at day 30 after cancer cell transplantation. Tumor xenografts, lungs, and livers of the mice were dissected; the tissues were analyzed by immunohistochemical staining and H & E staining. The remaining mice were euthanized by CO 2 asphyxiation on day 60 or when the xenografts reached 1.0 cm in diameter. Immunohistochemical staining of Ki-67 (CapitalBio, China), IL-8, and CD68 of tumor tissue sections was carried out according to the manufacturer's protocol. H&E staining of the lung and liver tissue sections was performed to examine pulmonary and hepatic metastasis. www.impactjournals.com/oncotarget In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 µg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control. The antibody injection was carried out when leptin or PBS injection started. Mice received 2 weeks of injection of leptin and the antibodies.