mRNA extraction from breast cancer tissue
The biospecimens including breast tissues (n = 90) and sera (n = 55), and the characteristic information of breast cancer patients used in this study were provided by the Biobank of Chonnam National University Hwasun Hospital (Hwasun-gun, Korea) and Korea University Guro Hospital (Seoul, Korea). RNA was extracted from frozen tissues using a homogenizer and TRI Reagent® (Ambion, Austin, TX), and then cDNA was synthesized using the M-MuLV reverse transcriptase (New England Biolabs, Beverly, MA) according to manufacturer’s instructions.
Cell culture and treatment
The MDA-MB-231 cell line (ATCC® HTB-26™, Manassas, VA) was cultured in DMEM (Hyclone Laboratories, Logan, UT). The human monocytic cell line THP-1 (Korean Cell Line Bank, Seoul, Korea, KCLB-40202) was cultured in RPMI-1640 (HyClone). Both mediums were supplemented with 10% heat-inactivated fetal bovine serum (MilliporeSigma, Burlington, MA), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C/5% CO2. To generate the conditioned media (CM), THP-1 cells were stimulated with 100 nM phorbol ester (PMA) (MilliporeSigma) for 48 h, the non-attached cells were washed with phosphate buffered saline (PBS) followed by addition of fresh culture media, and these cells were then incubated for another 24 h. The CM was collected and centrifuged to remove the remaining cells.
Generation of the IL-32θ-overexpressing cell line
We transfected MDA-MB-231 cells with the pcDNA3.1 (+)-6 × Myc-IL-32θ vector or pcDNA3.1 (+)-6 × Myc-empty vector, as described previously  and refer as MDA-MB-231-IL-32θ and MDA-MB-231-EV cells, respectively. In brief, the cells were seeded into 6-well plates (1 × 105 cells/well) and transfected with 3 μg of vector using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA). Afterwards, the cells were selected using medium containing 700 μg/ml G-418 (Duchefa Biochemie BV, Haarlem, The Netherlands) for two weeks. G-418-resistant colonies were then pooled and expanded.
Migration and invasion assays
For the migration assay, cells (5 × 105 cells/mL) were seeded onto the upper chambers of 24-well transwell plates (Corning Inc., Corning, NY) in serum-free DMEM. For the invasion assay, each transwell chamber was coated with 30 μl of Matrigel (Corning) before breast cancer cells were added to the top chamber. The lower chamber contained 500 μl macrophage-derived CM. Cells migrated or invaded for 24 h at 37 °C/5% CO2, and non-migrated or non-invaded cells were removed from the chamber interior by a cotton swab. Attached cells to the lower surface of the chamber were stained using Diff-Quick Kit (Sysmex, Kobe, Japan). Migrated or invaded cells were quantitated by dissolving stained cells in 100 μl of 10% acetic acid and then the mixture was transferred to a 96-well plate for colorimetric reading at 620 nm.
Quantitative reverse transcription PCR (RT-qPCR)
The mRNA expression levels in breast cancer cells were detected by RT-PCR for IL-32θ and RT-qPCR for other target genes. Total RNA was isolated using Easy-BLUE (iNtRON Biotechnology, SungNam, Korea), then reverse transcription was performed. qPCR was conducted using SensiFAST™ SYBR NO-ROX Kit (BIOLINE, London, UK). Samples were analyzed using the primer sets listed in Additional file 1: Table S1. Transcript levels were quantitated using the -ΔCt method (Ct = fluorescence threshold value; −ΔCt = Ct GAPDH – Ct target gene).
Enzyme-linked immunosorbent assay (ELISA)
Cells were cultured in the absence or presence of CM for 24 h, and then the culture media were replaced by fresh media for another 24 h. The cell culture supernatants were collected and analyzed using ELISA kits (R&D Systems, Minneapolis, MN) for human IL-1β, CCL5, CCL18, GM-CSF according to manufacturer’s instructions.
Immunoblotting and immunoprecipitation
For nuclear and cytoplasmic fractionation, cells were collected and fractionated using the NE-PER kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. For immunoprecipitation, cell lysates were mixed with specific antibodies and then pulled down by protein G-agarose beads. Samples were subjected to 10% SDS–PAGE before being transferred to PVDF membranes (MilliporeSigma). The membranes were blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.05% Tween-20 followed by primary antibody incubation at 4 °C overnight. After washing, horseradish peroxidase-conjugated IgG antibodies were added, and the membranes allowed to incubate for 1 h. Western blot was visualized using a chemiluminescence detection kit (Advanstar, Cleveland, Ohio) and detected by EZ-capture MG protein imaging system (ATTO, Tokyo, Japan). Specific antibodies used include those against Myc-tag, Flag-tag and phosphotyrosine-STAT3 (MilliporeSigma); IκBα, p-IκBα, p65, p50, PARP, and E-cadherin (Cell Signaling Technology, Danvers, MA); STAT3, COX-2, GAPDH (Santa Cruz Biotechnology, Dallas, TX); and anti-CCL18 neutralizing antibody (Abcam, Cambridge, MA). The monoclonal antibody KU-32-52 to detect IL-32 was prepared as previously described . The raw data of western blot results can be seen in Additional file 2.
Cells (3 × 105 cells/well) were seeded in a 6-well plate, cultured overnight, and then treated with or without CM for 24 h. MMP-9 activity in the supernatant was assayed as previously described . Gel staining was conducted with InstantBlue™ (MilliporeSigma) for 30 min in the dark. Areas of gelatinolytic degradation appeared as transparent bands on the blue background.
Cells were seeded on coverslips and incubated overnight. The attached cells were fixed, and permeabilized with cold acetone before blocking with 0.1% bovine serum albumin in PBS at room temperature (RT). Primary antibodies were added (1:100) to the coverslip incubating at 4 °C overnight. After washing with PBS, the coverslips were incubated with secondary antibodies at (1:200). Nuclei staining was performed by exposing to 4, 6-diamidino-2-phenylindole (1:2000) (MilliporeSigma) for 20 s. The stained cells were visualized using an upright fluorescence microscope (Olympus, Tokyo, Japan).
Xenograft model and optical imaging
All animal procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC No. KU17008) of Konkuk University. MDA-MB-231-EV and MDA-MB-231-IL-32θ cells (5 × 106 cells) were subcutaneously injected with Matrigel into the flanks of 5-week-old female athymic BALB/c nude mice (Nara Bio, Seoul, Korea). After 35 days, the tumors were harvested from euthanized mice. The tumor tissues were fixed with 10% formalin buffer, embedded in paraffin, and sectioned in 3 μm thickness for use in immunohistochemical analyses. Tumor volume was calculated using the formula V(mm3) = (shortest side2 × longest side)/2. For pre-and intra-operative tumor localization in the real-time resection, we conducted an in vivo tumor localization assay using the IRDye®-800CW 2-DG (2-deoxy-D-glucose) optical probe (LI-COR Biosciences, Lincoln, NE). Tumor localization was detected using optical imaging, particularly in the near-infrared fluorescence range. The tumorigenicity of MDA-MB-231-EV and MDA-MB-231-IL-32θ cells was assayed by intravenous injection of 1 × 106 cells resuspended in PBS into nude mice (n = 5 per group).
Formalin-fixed, paraffin embedded tumor tissue sections from mice were immersed in citrate buffer and boiled for 4 min in a microwave to retrieve antigens. Endogenous peroxidase activity was blocked with 3% (v/v) H2O2 for 10 min. Non-specific binding sites were blocked with 1% BSA for 30 min. Sections were incubated with the appropriate primary antibodies at 4 °C overnight and then the appropriate secondary antibodies for 1 h at RT. Diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA) was used as a substrate, and the sections were then counterstained with hematoxylin (MilliporeSigma).
Chi-square or Fisher’s exact test was used to evaluate the relationship between IL-32θ expression and clinicopathological status. The mRNA expression in tumor tissues and protein secretion in breast cancer patients’ sera were analyzed by Mann-Whitney U test. Student’s t-test were used to compare the two groups in in vitro and in vivo experiments. Statistical analyses were performed using GraphPad Prism software version 5.0. All p-values were two-sided, and p < 0.05 was interpreted as being statistically significant.