Paraffin-embedded samples were prepared from specimens obtained from 231 patients diagnosed with lung adenocarcinoma between 2010 and 2013 at Kumamoto University Hospital. Two pathologists reviewed all tissue specimens, and the most representative area of a 5-mm-diameter core containing viable lung adenocarcinoma cells was carefully selected for tissue microarrays. The study design was approved by the Kumamoto University Review Board (approval #1174).
The DAKO automation system (Autostainer Link 48; DAKO, Glostrup, Denmark) was used for the immunohistochemical analysis of human PD-L1 (clone 22C3; DAKO). Anti-human CD8 antibody (clone C8/144B; Nichirei, Tokyo, Japan), anti-PU.1 antibody (clone EPR3158Y; Abcam, Cambridge, UK), and anti-Iba-1 antibody (Wako, Tokyo, Japan) were used as the primary antibodies to label the macrophages and lymphocytes in human samples. For IHC of murine tumor specimens, anti-Iba-1 antibody (Wako), anti-CD8 antibody (clone D4W2Z; Cell Signaling Technology, Danvers, MA), and anti-PD-L1 antibody (#AF1019; R&D Systems, Minneapolis, MN, USA) were used as the primary antibodies. Horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin antibody (Nichirei) was used as the secondary antibody. 3,3’-Diaminobenzidine was used for the visualization of positive signals in the first step of double IHC. Subsequently, sections were treated by heating in 1 mM ethylenediaminetetraacetic acid (pH 8.0) buffer. Then, the sections were treated with HRP-labeled anti-rabbit immunoglobulin antibody, and positive signals were visualized with HistoGreen substrate (#AYS-E109; Linaris, Dossenheim, Germany) as the second step of double IHC. Two investigators (Y.K. and Y.S.), who were blinded to information about the samples, evaluated the PD-L1 and PU.1 expression. We also determined the macrophage proportion score (MPS), which is based on the tumor proportion score (TPS). Images of ten randomly selected 400 × fields were obtained under microscopy, and the image files were analyzed for cell counting and the evaluation of the stained areas by Image J software.
Cell culture of macrophages, cancer cell lines, and lymphocytes
Monocytes were isolated using RosetteSep Human Monocyte Enrichment Cocktail (STEMCELL Technologies, Vancouver, Canada). Peripheral blood mononuclear cells were obtained from three healthy voluntary donors in accordance with protocols approved by the Kumamoto University Hospital Review Board (#1169). These monocytes were plated on UpCELL 6-well plates (2 × 105 cells/well; CellSeed, Tokyo, Japan) and cultured in AIM-V medium (Thermo Fisher, Waltham, MA, USA) supplemented with 2% human serum macrophage-colony stimulating factor (M-CSF; 100 ng/mL; Wako) for 7 days to induce the differentiation of macrophages.
Three human lung adenocarcinoma cell lines (NCI-H23, H358, and H1975) were obtained from Tomoya Yamaguchi (Kumamoto University, Kumamoto, Japan). A549 and PC9 were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Murine Lewis lung carcinoma (LLC) cells were kindly gifted from Keizo Takenaga (Chiba Cancer Center, Chiba, Japan). All cells were cultured in RPMI1640 (Wako) supplemented with 10% fetal bovine serum. The conditioned medium (CM) of the cell lines was collected as previously described . For the cell culture study using mouse macrophages, bone marrow cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum and M-CSF (100 ng/mL) for 5 days, and adherent cells were used as macrophages.
Lymphocytes, isolated from healthy donors using RosetteSep Human T-cell Enrichment Cocktail (STEMCELL Technologies), were cultured in a cell-culture plate coated with anti-human CD3 antibody (OKT3; eBiosciences, San Diego, CA, USA), human CD28 antibody (BioLegend, San Diego, CA, USA). The proliferation of lymphocytes was tested by BrdU incorporation assay (Cell proliferation ELISA kit, Roche, Basel, Switzerland). Anti-PD-1 antibody (clone EH12.2H7) and isotype matched IgG were obtained from BioLegend (San Diego, CA, USA).
Cell enzyme-linked immunosorbent assay (ELISA)
Macrophages were cultured in a 96-well microplate and stimulated with the CM of the adenocarcinoma cell lines for 1 day. After fixation with 1% paraformaldehyde, cells were reacted with anti-PD-L1 antibody (clone 29E.2A3; BioLegend, San Diego, CA, USA) or isotype-matched control antibody (BioLegend). After the cells were washed with phosphate-buffered saline, HRP-labeled anti-mouse immunoglobulin antibody (Nichirei) was added. Then, the plate was washed with phosphate-buffered saline, and tetramethylbenzidine developing solution (BioLegend) was used to visualize the positive signals.
Phospho-receptor tyrosine kinase (RTK) array
Phospho-RTK array analysis was performed using the Human Phospho-RTK Array Kit (ARY 001; R&D Systems) according to the manufacturer’s instructions.
Cytokine array analysis was performed using the Human XL Cytokine Array Kit (ARY 022; R&D Systems) according to the manufacturer’s instructions.
ELISA for granulocyte–macrophage colony-stimulating factor (GM-CSF)
ELISA for GM-CSF was performed using the Human GM-CSF ELISA Kit (Cat. No. 432007; BioLegend) according to the manufacturer’s instructions.
Recombinant proteins, anti-cancer chemicals, and inhibitors
IL-6 and GM-CSF recombinant proteins were purchased from Wako. The following inhibitors were used at a final concentration of 10 nM: Stat1 (Fludarabine; Wako), Stat3 (WP1066; Santa Cruz, Dallas, TX, USA), Stat5 (573,108; Merck KGaA, Darmstadt, Germany), JNK (SP600125; Santa Cruz), ERK (FR180204; Santa Cruz), and JAK (Ruxolitinib; ChemScene LLC, Monmouth Junction, NJ, USA). Paclitaxel, docetaxel, carboplatin, and pemetrexed were obtained from Wako.
Western blot analysis
The macrophages were stimulated with the CM of the lung adenocarcinoma cell lines (concentration: 50%) for 10 min, 30 min, 1 h, 3 h, or 1 day. Then, the macrophages were collected, and the cellular proteins were solubilized in Tris buffer containing 2% sodium dodecyl sulfate and 10% glycerol. The amount of protein was quantified using the bicinchoninic acid assay. Equal amounts of protein were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene fluoride membrane. The following rabbit antibodies were used for western blotting: anti-PD-L1 antibody (clone E1L3N; Cell Signaling Technology), anti-STAT3 antibody (clone 124H6; Cell Signaling Technology), and anti-pSTAT3 antibody (clone Y705; Cell Signaling Technology).
Human monocyte-derived macrophages were treated with human FcR-blocking reagent (BioLegend) and then reacted with phycoerythrin-labeled or Alexa 488-labeled anti-human PD-L1 antibody (BioLegend) or isotype-matched control antibody (BioLegend). For analysis of murine subcutaneous tumor, anti-CD11b antibody, anti-PD-L1 antibody, and isotype-matched antibodies (BioLegend) were used. The stained cell samples were analyzed on a FACSverse (Becton Dickinson, Franklin Lake, NJ, USA) flow cytometer with FACSuite (Becton Dickinson) software.
C57BL/6 J mice were obtained from CLEA Japan (Shizuoka, Japan). LLC cells (5 × 105 cells/mouse) suspended in 50 µL of RPMI1640 medium were subcutaneously injected into the mice. Anti-GM-CSF antibody (clone MP1-22E9) and isotype-matched control antibody were purchased from BioXel (New Haven, CT, USA). Hamster anti-mouse PD-L1 antibody (clone 10B5; 100 mg/mice) was established previously , and control hamster immunoglobulin G was obtained from Sigma (St. Louis, MO, USA). All animal experiments were approved by the Ethics Committee for Animal Experiments of Kumamoto University (#A2019-176) and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee.
Statistical analysis was carried out using GraphPad Prism9 (https://www.graphpad.com/) and JMP7 (SAS Institute, Chicago, IL, USA) software. Differences were considered to be statistically significant at p < 0.05.