Isolation and culture of peripheral blood mononuclear cells (PBMCs)
PBMCs were collected from six lung cancer patients and four healthy subjects. This study was approved by the Taipei Medical University Joint Institutional Review Board, and informed consent was obtained from all subjects. All experiments were performed in accordance with relevant guidelines and regulations. Among the lung cancer patients (Supplementary Table S1), five had adenocarcinomas (83.3%) and one patient was diagnosed with squamous cell carcinoma (16.7%).
Venous blood was drawn and buffy coats were obtained through separation using SepMate tubes (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. In brief, heparinized blood was diluted with phosphate-buffered saline (PBS), layered on top of Lymphoprep, and centrifuged at 1200×g for 10 min. PBMCs were collected, washed twice with PBS, and counted using a hemocytometer with trypan blue (Lonza, Basel, Switzerland) to determine cell viability. PBMCs were cultured in the same conditions as THP1 and A549 cells in the following section. After 24 and 72 h of culture, cells were washed, and adherent cells were collected to observe macrophage polarization using a flow cytometric assay.
THP1-derived classically activated macrophages (Ms) and tumor-educated macrophages (TEMs)
An in vitro study was developed utilizing the THP1 human monocytic cell line (ATCC® TIB-202™, ATCC, Manassas, VA, USA) and the A549 lung adenocarcinoma cell line (ATCC® CCL-185™) for macrophage polarization, cytokine, and cell viability assays. Approximately 2 × 106 THP1 and A549 cells were seeded in 10-cm dishes in filter-sterilized RPMI-1640 medium, 10% heat-inactivated fetal bovine serum (Gibco, Waltham, MA, USA), and 100 U/mL penicillin–streptomycin (Gibco). Cells were grown in a humidified incubator at 37 °C under 5% CO2. Routine subculturing was conducted every 3 days. Cells between passages two and eight were used for experiments. For differentiation, 2 × 106 THP1 cells were seeded in 10-cm dishes and treated with 80 nM phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, St. Louis, MO, USA) for 24 h. For Ms, culture media were replaced with RPMI-1640 media and incubated for 24 h. To prepare TEMs, THP1-derived macrophages were further co-cultured with A549 cells in 0.4-µm polycarbonate permeable trans-well dishes (Corning, Corning, NY, USA) for 24 h.
Heat-killed tuberculosis (HKTB) stimulation
Heat-killed Mycobacterium tuberculosis (HKTB) avirulent H37Ra strain (InvivoGen, San Diego, CA, USA) was prepared according to manufacturer’s instructions and used to stimulate Ms and TEMs, either in transwell chambers or tissue culture dishes, for 24 or 72 h in a humidified incubator at 37 °C under 5% CO2. Prior to harvesting, cells were washed with PBS and adherent cells were collected.
Flow cytometry
Human peripheral blood macrophages and THP1-derived macrophages were submitted to flow cytometric analysis according to the manufacturer’s recommendations. After being washed with PBS, cells were stained with a LIVE/DEAD™ fixable blue dead cell stain kit (Invitrogen, Paisley, UK), followed by blocking of Fc receptors using Human TruStain FcX (BioLegend, San Diego, CA, USA) and cell surface labeling by a specific antibody or isotype control. Antibodies against CD11b, CD86, CD206, CD14, and human leukocyte antigen (HLA)-DR were used for the human peripheral blood macrophages, while antibodies against CD86, CD206, CD14, HLA-DR and CD68 were used for THP1-derived. All these antibodies were purchased from BioLegend (San Diego, CA, USA). Samples were washed once and analyzed in a BD LSRFortessa cytometer (BD Bioscience, San Jose, CA, USA) and FlowJo™ (v10, LLC, Ashland, OR, USA). Results are presented as the percentage of positive cells or as the ratio of the mean of fluorescence intensity (MFI) of the antibody of interest to the isotype control. Three technical replicate analyses were performed for human peripheral blood macrophages, while three biological and three technical replicate analyses were performed for THP1-derived macrophages.
Tandem mass tag (TMT)-based quantitative proteomics analysis
THP1-derived M and TEM cells and the HKTB-stimulated M (TB-M) and TEM (TB-TEM) cells were first washed three times with PBS and scraped into lysis buffer containing 6 M urea, 5 mM ethylenediaminetetraacetic acid (G-Biosciences; St. Louis, MO, USA), 2% sodium dodecylsulfate, and 0.1 M triethylammonium bicarbonate buffer (TEABC, Sigma Aldrich, St. Louis, MO, USA). A protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) was added in a volume ratio of 100:1 (sample: protease inhibitor, v/v, Calbiochem), followed by sonication at 4 °C for 15 min (Bioruptor, Diagenode, Belgium) and 30 min of centrifugation at 13,000×g. The supernatant was collected and quantified using a Pierce Bicinchoninic Acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL, USA) to determine the protein concentration. Fifty micrograms of proteins were subjected to our previously reported gel-assisted digestion55. The resulting peptide was concentrated in a SpeedVac (Thermo Fisher Scientific) and resuspended in 100 mM TEABC for the BCA assay. Five micrograms of peptides from each sample were taken for labeling with TMT isobaric reagents (Thermo Fisher Scientific) following the manufacturer’s instructions. Untreated and HKTB-treated Ms were labeled with TMT127 and TMT128, respectively, while untreated and HKTB-treated TEMs were labeled with TMT129 and TMT130, respectively.
TMT-labeled peptides were pooled and fractionated using high-pH reverse-phase StageTip to collect six fractions following a previous study56. Each fraction was desalted by C18 ZipTip (Millipore, Cambridge, Ontario, Canada) and resuspended in mobile phase buffer A (0.1% formic acid in H2O) for subsequent LC–MS/MS analysis on a nanoAcquity system (Waters, Milford, MA, USA) connected to an LTQ-Orbitrap Velos (Thermo Fisher Scientific) equipped with a Nanospray Flex interface. Briefly, peptide mixtures were loaded onto a 75-μm i.d. × 25-cm C18 BEH column (Waters) packed with 1.7-μm particles with a pore size of 130 Å and separated using a segmented gradient from 1 to 45% of mobile phase buffer B (0.1% formic acid in acetonitrile) for 103 min at a constant flow rate of 0.3 µL/min and a column temperature of 35 °C. Peptides were detected in the data-dependent acquisition mode. Full-scan MS spectra were acquired in the orbitrap (m/z 350 to 1600, resolution 60,000 at m/z 400). The top 10 most intense ions with at least two positive charge states were sequentially isolated (with an isolation window of 2 Da and automatic gain control of 5E5) and fragmented by high-energy collision-induced dissociation in a multipole collision cell with a normalized collision energy of 45%. Fragmented ions were detected in the linear ion trap to acquire the MS/MS spectra.
For the proteomic analysis, three independent biological replicates and two technical replicates were analyzed, and proteins were identified by searching MS raw files against the SwissProt human protein sequence database (release 2019_02, 20,335 entries) using Mascot implemented in Proteome Discoverer (vers. 2.2.0.388, Thermo Fisher Scientific). Only tryptic peptides with up to two missed cleavages were allowed. The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.1 Da, respectively. Methylthio (Cys) was set as a fixed modification, whereas oxidation (Met), acetylation (protein N-terminal), deamidation (Asn, Gln), and TMT tags (N-terminal, Lys) were set as variable modifications. Peptide-spectrum matches were validated using percolator (q-value of 1%). Peptide-spectrum matches and proteins with a < 1% false discovery rate were considered positive identifications.
For proteome quantitation, only master proteins with at least one unique peptide were quantified, and protein ratios were normalized by total peptide abundances. Based on our previous study57, we considered proteins with log2 ratios (TB-M/M or TB-TEM/TEM) of > 0.3785 or < -0.3785 (indicating a 1.3-fold difference in abundance) as differentially expressed proteins (DEPs).
Functional enrichment analysis of DEPs
DEPs identified in at least two biological replicates were submitted to Gene Ontology (release 2019-06-01)58,59 for enrichment of dysregulated biological processes and to Ingenuity Pathway Analysis (IPA)60 for pathway enrichment analysis. Z-scores were obtained from the IPA for enriched canonical pathways and biofunctional annotations. Only annotations with p < 0.05 were considered significant hits. A z-score of > 0 indicates activation, while one of < 0 indicates inhibition of a cellular function or pathway. Unsupervised hierarchical clustering of proteome expression profiles was performed with Gene Cluster 3.0 vers. 1.5261, and visualized with Java Treeview vers. 1.1.6r462.
Western blot analysis
Twenty-μg of protein was run in gel electrophoresis by using 4–20% Mini-PROTEAN® TGX™ Gel (Bio-Rad, CA, USA), and transferred onto a nitrocellulose membrane. The membranes were blocked for one hour at room temperature and incubated overnight at 4 °C with primary antibodies against Nfkb p65, Stat1, and Gapdh (Abcam, MA, USA) at a:1000 concentration. Following three times washing step using phosphate-buffered saline with Tween-20, the membranes were incubated with the antirabbit or antimouse IgG secondary antibody in a concentration of 1:10,000 (Bioss Antibodies, MA, USA) for 1 h at room temperature. Clarity™ Western ECL Substrate was used to detect protein bands. Gapdh was used as the loading control, and three replicates of experiments were performed to obtain statistical significance. The detected protein bands were quantified using AzureSpot (Azure Biosystems, CA, USA).
Quantification of cytokine levels by an enzyme-linked immunosorbent assay (ELISA)
Cytokine levels in culture media were quantified using an ELISA Ready-SET-Go kit (Ebioscience, San Diego, CA, USA) according to the manufacturer’s instruction. Proinflammatory tumor necrosis factor (TNF)-α, IFN-γ, and interleukin (IL)-1β cytokines and anti-inflammatory IL-4, IL-10, and transforming growth factor (TGF)-β cytokines were assayed. Samples were run in triplicate and quantified using a standard curve.
Inhibition of IFN-γ and TNF-α
Neutralization antibodies against IFN-γ (20 µg/mL; Ebioscience, San Diego, CA, USA) and TNF-α (10 µg/mL; R&D Systems, Minneapolis, MN, USA) were added to THP1-derived macrophages for 1 h, followed by 72 h of HKTB stimulation. Pre-stimulated macrophages were then cocultured with A549 cells to determine their cytotoxic capability.
Cell viability assay of A549 cells
A549 cells (105) were seeded in the bottom chamber of six-well plates and cultured overnight. HKTB-stimulated or non-stimulated macrophages were pre-prepared in 0.4-µm polycarbonate permeable trans-well dishes (Corning) and respectively cocultured with A549 cells for 24 and 72 h. A549 cell viability was determined using an MTT Cell Growth Assay Kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. To determine the cell death profile, A549 cells were also subjected to staining with a FITC Annexin V Apoptosis Detection Kit with propidium iodide (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. At least three replicates of samples were submitted for each assay.
Statistical analysis
Data obtained from macrophage polarization experiments and cytokine assays were analyzed by Student’s t-test. Cell viability results were analyzed by a two-way analysis of variance (ANOVA) followed by a post-hoc multiple-comparison test. Data graphs are presented in mean values with standard deviations and generated with GraphPad Prism (La Jolla, CA, USA). For all statistical analyses, results with p < 0.05 were considered significant.
