Patients
A total of 145 formalin-fixed paraffin-embedded (FFPE) tissue samples of breast cancer were obtained from Renmin Hospital of Wuhan University. All patients involved in the study provided a written informed consent, and the study was approved by the Institutional Ethics Committee of Renmin Hospital of Wuhan University (approval no. 2018K-C09). Patients did not receive financial compensation. Clinical information was extracted from medical records and pathology reports, and the detailed clinicopathological characteristics of the patient are shown in Table 1. Patients were all followed-up for at least 5 years from the date of first diagnosis. All methods were performed in accordance with relevant guidelines and local regulations.
Cell culture and reagents
The breast cancer cell lines AT3, 4T1, MCF-7, MDA-MB-231 and monocyte lines Raw264.7, THP-1 cells were obtained from American Type Culture Collection (ATCC, Shanghai). The mouse breast cancer cell line 4T1 and human monocyte line THP-1 were cultured in Roswell Park Memorial Institute (RMPI)-1640 medium supplemented with 10% foetal bovine serum (FBS, AusGeneX) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA) in a humidified 37 °C incubator with 5% CO2. The human breast cancer cell lines MCF-7, MDA-MB-231 and mouse macrophage line Raw264.7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin–streptomycin in a humidified 37 °C incubator with 5% CO2. 3T3-L1 preadipocytes were obtained from ATCC (Shanghai) and cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin in a humidified 37 °C incubator with 5% CO2. Cytochalasin D, Bafilomycin A1(Baf-A1) and insulin were purchased from Sigma.
Coculture and invasion assays
Mature 3T3-L1 and breast cancer cells were cocultured using Transwell culture plates (0.4μm pore size; Millipore). Mature 3T3-L1 cells in the bottom chamber of the Transwell system were cultivated in serum-free medium containing 1% bovine serum albumin (Sigma) for 4 h. A total of 3 × 10^5 4T1 or MCF-7 or MDA-MB-231 cells were cultivated in the top chamber in the presence or absence of mature 3T3-L1 cells in the bottom chamber for the indicated times. The conditioned medium (CA-CM) was collected from adipocytes cultivated with tumour cells for 3 days. After 24 h of coculture in the presence of normal medium or CA-CM (supplemented with 10% FBS), macrophages were seeded in the top chamber in the presence of CM, AD-CM or CA-CM (supplemented with 10% FBS) for Matrigel invasion assays.
THP-1 Differentiation
A total of 5 × 10^6 THP-1 cells were seeded in a 6-well plate with Phorbol 12-myristate 13-acetate (PMA, 100ng/ml, MedChemExpress, 16561-29-8) supplemented with 1% FBS in RMPI-1640. After 24h, RMPI-1640 medium with PMA was replaced by normal medium for 2 days.
Exosome isolation and characterization
After cells were cultured with exosome-depleted serum (AusGeneX), the exosomes were purified from the conditioned medium according to the instructions[23]. The medium was centrifuged at 500 g for 5min and at 2000 g for 30min at 4 °C to remove cellular debris and large apoptotic bodies. After centrifugation, media was added to an equal volume of a 2× polyethylene glycol (PEG, MW 6000, Sigma, 81260) solution (final concentration, 8%). The samples were mixed thoroughly by inversion and incubated at 4 °C overnight. Before the tubes were tapped occasionally and drained for 5min to remove excess PEG, the samples were further centrifuged at maximum speed (15,000 rpm) for 1 h at 4 °C. The resulting pellets were further purified using 5% PEG and then stored in 50–100 μl of particle-free PBS (pH 7.4) at − 80 °C. The average yield was approximately 300 μg of exosomal protein from 5 ml of supernatant. Total RNA was extracted by using Trizol reagent (Life Technologies), followed by miRNA assessment by microarrays and RT-PCR described below. Exosomes were analyzed by electron microscopy to verify their presence, by a nanoparticle characterization system to measure their size and concentration, and by western blot to detect their proteins (TSG101, CD63 and CD81).
Electron microscopy
After being fixed with 2% paraformaldehyde, samples were adsorbed onto nickel formvar-carbon-coated electron microscopy grids (200 mesh), dried at room temperature, and stained with 0.4% (w/v) uranyl acetate on ice for 10 min. The grids were observed under a HITACHI HT7700 transmission electron microscope.
Nanoparticle characterization system (NanoSight)
The NanoSight (Malvern Zetasizer Nano ZS-90) was used for real-time characterization and quantification of exosomes in PBS as specified by the manufacturer’s instructions.
Exosome uptake analysis
Exosomes derived from breast cancer cells were labeled by the cell membrane labeling agent PKH26 (Sigma-Aldrich). After being seeded in 96-well plates and allowed to differentiate, mature 3T3-L1 cells were incubated with labelled exosomes (20 μl/well) for the indicated time. Images were acquired using the Olympus FluoView FV1000.
ELISA
The conditioned media were collected to analyze the secretion levels of CCL2 and CCL5 via Mouse MCP-1/CCL2 ELISA kit (Sizhengbai, CME0046) and Mouse CCL5/RANTES ELISA kit (Sizhengbai, CME0048). The conditioned media were centrifuged at 1000 g for 10min, incubated with biotinylated antibody working solution at 37℃ for 1.5h, incubated with enzyme conjugated working solution at 37℃ for 0.5h, and color developing for 10min.
Western blotting
After being washed twice with ice-cold PBS, cells were collected with SDS loading buffer and boiled for 10 min. The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with specific antibodies (Additional file 1: Table S1).
Neutralizing experiments
Before used to treat macrophages, CA-CM was neutralized by goat serum or CCL2 or CCL5 neutralizing antibodies overnight. Macrophages were treated with the neutralized CA-CM for 3 days, and then were collected for western blot analysis.
RNA extraction and quantitative PCR
Gene expression was analyzed using real-time PCR. The mRNA primer sequences are provided in Additional file 1: Table S2. The miRNA primer kits were purchased from RiboBio (Guang Zhou, China).
Immunohistochemistry
A cohort of 145 human breast cancer specimens was collected from Renmin Hospital of Wuhan University from 2011 to 2013. Immunohistochemistry (IHC) staining was performed, and the staining results were scored by two independent pathologists based on the proportion of positively stained tumour cells and the staining intensity. The protein expression level of CD163 was described according to the numbers of CD163+ macrophages counted in 10 random fields of each breast cancer specimen at 400× magnification. The intensity of protein expression was scored as 0 (no staining), 1 (weak staining, light brown), 2 (moderate staining, brown) and 3 (strong staining, dark brown). The protein staining score was determined using the following formula: overall score = percentage score × intensity score. Receiver operating characteristic (ROC) analysis was used to determine the optimal cut-off values for all expression levels regarding the survival rate.
Immunofluorescence imaging
Immunofluorescence (IF) imaging was performed to investigate the localization of pSTAT3 (Tyr705) (#9145, 1:200, Cell Signaling Technology), CD68 and CD163. Tissue specimens undergoing IF staining were incubated with Alexa Fluor -conjugated secondary antibodies against the primary antibodies for 1 h at room temperature, followed by counterstaining with DAPI for 5 min. Images were captured using a fluorescence microscope (Olympus BX63; Olympus Corporation).
Image segmentation and data analysis
Images were segmented using the EBImage package (available from Bioconductor repository https://www.bioconductor.org) with the R software. The nuclear region was defined using a polygon mask based on the nuclear Hoechst signal, and a second polygon mask was generated using the GFP or RFP signal. For the assessment of autophagic vesicles, a third mask was created on cytoplasmic regions exhibiting a high intensity signal of GFP corresponding with LC3 aggregates.
Luciferase assays
The 3’ UTRs of target genes containing predicted miRNA binding sites (genewt) were cloned into the GV272 vector (GeneChem Biotechnology, Shanghai, China), and the miRNA binding sites were replaced with a 4-nt fragment to produce a mutated 3’ UTR (genemut) in the vector. Briefly, HEK 293 T cells were seeded in 12-well plates and grew to 70% confluence. The cells were transfected with genewt or genemut, the pre-miRNA expression plasmid and pRL-SV40, which constitutively expresses Renilla luciferase as an internal control. At 48 h post-transfection, the cells were lysed, and Renilla luciferase activity was assessed by the TECAN Infiniti reader. The results are described as the ratio of firefly luciferase activity compared to Renilla luciferase activity.
Lentivirus preparation and transfection
siRNA-TFE3 (5’-GGAAUCUGCUUGAUGUGUA-3’) (GE Healthcare, Dharmacon) were used. LentiBrite GFP-LC3 lentiviral biosensor was purchased from MERCK (#17-10193, Darmstadt, Germany). miRNA-155 inhibitors and pre-miRNA lentivirus were obtained from GeneChem Biotechnology (Shanghai, China). Cells were cultured at 5 × 105 cells/well in 6-well plates. After being incubated for 24 h, the cells were transfected with siRNA lentivirus and control sequences using CON036 (GeneChem Biotechnology, China) following the manufacturer’s instructions. Cells (2 × 105) were stably transfected with empty vector or with vectors carrying miRNA inhibitor or pre-miRNA using the TransIT-LT1 reagent (Mirus). Selection was carried out with puromycin (1 μg/ml, Sigma) or G418 (500 μg/ml, Sigma) in cell culture media for 48 h after transfection. Selected clones were maintained in DMEM with 500 μg/ml G418 or 1 μg/ml puromycin. Cell lysates were collected, and RT-PCR was performed to detect miRNA expression. The sequence information is provided in Additional file 3: Table S3.
Xenograft tumor formation
Six-week-old female BALB/c mice were purchased from Vital River, Beijing. The animals were handled according to the protocol approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University. The following cell lines were used to create subcutaneous models: 4 × 105 4T1 cells treated with control lentivirus, transfected with miRNA-155 inhibitor lentivirus. Breast cancer cells were injected alone or in combination with mature adipocytes (1 × 105 cells). All cell samples were subcutaneously injected with Matrigel (1:1), total volume 100 μl, into the axilla of the mice. For macrophages deletion, mice were treated with blocking antibodies against F4/80 as described.
Six-week-old female wild-type C57BL/6 mice were obtained from Vital River, Beijing. Mouse Mammary Carcinoma AT-3 cells (2 × 105) alone or in combination with mature adipocytes (1 × 105 cells) were subcutaneously injected into C57BL/6 hosts. When tumors became palpable, mice were treated with the STAT3 inhibitor Stattic (10 mg/kg, days 0 and 7, Sigma-Aldrich), blocking antibodies against CCL2 (BioXcell, West Lebanon, NH, USA) and/or CCL5 (200 μg, days −1 and 0, and were repeated every 3 d thereafter to maintain depletion or neutralization) (R&D Systems, Minnesota, USA) by intraperitoneal injection, or the CCR2/CCR5 antagonist BMS-813160 (10 mg/kg, gavage, days 0, 4, 8 and 12) (MedChemEcpress, Shanghai, China). On the following days, mice well-being and tumor growth were monitored and documented. Tumour volume was defined as (longest diameter) × (shortest diameter)2 ×0.52 and was measured once every 2-3 days until using a Vernier calliper. Animals were sacrificed when tumor size reached endpoint or signs of obvious discomfort were observed following our Ethical Committee advice. After the mice were sacrificed, all tissues were collected, embedded in paraffin and stained with IHC or haematoxylin-eosin (HE).
Statistical analysis
All experiments were done independently at least three times. The results are presented as the mean ± SD. The relative increase in protein expression was quantified using Image J software and was normalized to control protein expression in each experiment. Data sets obtained from different experimental conditions were compared with the t-test when comparing only 2 groups. Multiple comparisons between groups were performed using the Mann–Whitney U test or Tukey’s multiple comparisons. Survival probabilities for recurrence-free survival (RFS) were estimated using the Kaplan–Meier method, and variables were compared using the log-rank test. Pearson’s correlation was used to evaluate the correlations among CD163, CCL2 and CCL5 expression levels. In the bar graphs, a single asterisk (*) indicates P < 0.05.