Chemicals and reagents
The following compounds and reagents were used: niclosamide was purchased from Selleck Chemicals, USA, and prepared in dimethyl formamide (DMF) at a concentration of 5 mmol/L and maintained at − 20 °C. Niclosamide was diluted in DMF for working solutions and used at concentrations ranging from 0.25 μmol/L to 2 μmol/L for the treatment of cells. The following antibodies were purchased from Cell Signaling Technology, USA: phospho-STAT3 (p-STAT3), STAT3. Human B7H1/PD-L1 polyclonal antibody was purchased from Santa Cruz Biotechnology, USA, and GAPDH antibody was from Abcam, UK. Polyclonal goat anti-mouse antibody and goat anti-rabbit antibody (R&D systems, USA) were used for Western blotting.
Cell lines and cell culture
NSCLC cell lines (A549, H1299, H460), Lewis lung cancer (LLC) cell line, and 293 T cell line were obtained from the American Type Culture Collection (ATCC, USA) and validated by short-tandem-repeat (STR) analysis (except for LLC). Cells were cultured in either RPMI-1640 (for NSCLC cell lines) or DMEM (for LLC cells and 293 T cells) containing 10% fetal bovine serum and maintained at 37 °C in a humidified 5% CO2 incubator. Peripheral blood mononuclear cells (PBMCs) were cultured in T cell medium (RPMI-1640 supplemented with 10% human serum, 5% L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich, USA), and IL-2 (100 IU/mL).
Cell cytotoxic assay
Cytotoxicity studies were performed using the thiazolyl blue tetrazolium bromide (MTT) assay. MTT assay was performed to examine sensitivity of the cells to niclosamide as described previously . Experiments were conducted at least three times. The concentration of niclosamide suppressing cell proliferation by 30% (IC30), calculated from survival curves using the Bliss method, was selected for further experiments.
Western blot analysis
Cells were treated with the indicated concentrations as shown in the figures and washed twice with cold PBS. Whole cell extracts were collected in RIPA lysis buffer (Santa Cruz Biotechnology, Germany), and protein concentration of the lysates was measured using a BCA Protein Assay Kit (Pierce Biotechnology, USA). The protein samples were electrophoresed through a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking, membranes were probed with primary antibodies (1:1000) followed by washing and incubation with a secondary antibody (1:5000) conjugated to horseradish peroxidase (Amersham GE Healthcare, USA). Protein bands were visualized by applying a chemiluminescent reagent (Pierce ECL kit, Thermo Fisher Scientific, USA).
RNA extraction and quantitative real-time PCR
Total cellular RNA was isolated using Trizol (Life Technologies, USA) according to the manufacturer’s protocol. For first-strand cDNA synthesis, 5 μg of total RNA was reverse-transcribed using the GoScript™ Reverse Transcription System kit (Promega, USA) followed by quantitative polymerase chain reaction (qPCR) with GoTaq qPCR Master Mix (Promega, USA), according to the manufacturer’s instructions. Real-time PCR analyses were conducted using the Biorad CFX96 system with SYBR green (Bio-Rad, USA) and the appropriate primers to estimate the mRNA expression levels of STAT3 and PD-L1. Data were normalized to GAPDH levels. Experiments were performed in triplicates. The primes are as follows: Stat3 forward: CTTGACACACGGTACCTGGA; reverse: CTTGCAGGAAGCGGCTATAC; PDL1 forward: TATGGTGGTGCCGACTACAA; reverse: TGCTTGTCCAGATGACTTCG; β-actin forward: TCCTGTGGCATCCACGAAACT; reverse: GAAGCATTTGCGGTGGACGAT.
Transfection of shRNA and plasmid DNA
STAT3 shRNAs and a shRNA scramble control (Additional file 1: Table S1) (Open Biosystems GE Healthcare Dharmacon Inc., USA) were transiently transfected along with a pSIH-H1-puro Lentivector Packaging Kit (System Biosciences, USA). Transfections were carried out in 293 T cells grown to ∼80% confluency in 10 cm dishes using Lipofectamine 2000 transfection reagent (Life Technologies, USA) and following the manufacturer’s instructions. H460 and H1299 cells were infected and incubated with the viral particles overnight at 37 °C. At 48 h after transfection, cells were placed under puromycin selection by supplementing the growth medium with puromycin (3 μg/ml for H460, and 4 μg/ml for H1299). Stable repression of gene expression was verified by Western blotting and RT-PCR.
Dual-luciferase reporter assay
An 868-bp PD-L1 promoter fragment (UCSC: http://genome.ucsc.edu/, the gene ID: 29126) (nucleotides − 762 to + 106 base pair (bp) relative to the translation initiation site) was PCR-amplified from H460 cell line genomic DNA and inserted into the promoter-less plasmid pGL3-Basic (Promega, USA), designated as p868. A series of 5′-deletions were produced by PCR using p868 as a template with the distinct 5′ primers a common 3′ primer (Additional file 1: Table S2). The products were cloned into pGL3-Basic to generate p693, p516, and p360. The promoter sequences were then interrogated for transcription factor binding sites and transcription factor modules with the use of PROMO (http://alggen.lsi.upc.es/) and the JASPAR database (http://jaspar.genereg.net). The STAT3 cDNA was PCR amplified with the relevant primers (Additional file 1: Table S2) and cloned into the plasmid PCDNA3.1 (Promega, USA). The 293 T cell lines were grown to approximately 80% confluence, and 4 × 105 cells each were co-transfected with 3.8 μg/well of pGL3 luciferase construct (empty vector or pGL3-PD-L1promoter) and 0.2 μg/well pRL-TK (Promega, USA). The relative luciferase activity was examined by Dual Luciferase Assay Kit (Promega, Madison, WI, USA) in accordance with the manufacturer’s protocols.
Colony formation assay
As effector cells, human PBMCs were purified from the blood of healthy volunteers using Ficoll gradient centrifugation (Solarbio, Beijing). The purity of the isolated cells was > 95%, as determined in flow cytometry (FCM). Briefly, 24-well plates were coated overnight with 5 μg/ml anti-CD3 (BD Bioscience, USA), then washed twice with PBS. PBMCs were plated in complete TCCM medium (IMDM with human AB serum (5%), penicillin–streptomycin, HEPES, 2-mercaptoethanol, and gentamicin). As target cells, cancer cells were pre-treated with niclosamide (2 μmol/L) for 24 h; control cells were without niclosamide pre-treatment. Then, cells were treated with PD-L1 Ab or not and co-cultured with activated PBMCs at several target-to-effector ratios (1:0, 1:1, 1:4, 1:16) (all samples in triplicate). After 4 days of co-incubation, 24-well plates wells were washed with PBS twice to remove PBMCs and then the survived tumor cells were fixed and stained with Giemsa staining solution. The dried plates were scanned and quantified the intensity.
Flow cytometry analysis
6-well plates were coated overnight with 5 μg/ml anti-CD3 (Biolegend, USA), then washed twice with PBS. PBMCs were plated at a density of 1 × 106/well in 6-well plates and then co-cultured with tumor cells pre-treated with niclosamide at 4:1 ratio for 24 h. Anti-human PD-L1 antibody, atezolizumab (Selleck Chemicals, USA) (50 μg/ml), was added to the appropriate wells. After co-culturing, the PBMCs were isolated and stained with anti-CD3 and anti-CD8 antibodies to estimate the CD8+ cell proportions. For TNF-α and granzyme B analysis, PBMCs were harvested and then treated with brefeldin A (Biolegend, USA) at 37 °C for an additional 3 h to prevent extracellular secretion. Subsequently, PBMCs were fixed and permeabilized with the Intracellular Fixation and Permeabilization Buffer Set Kit (eBioscience, USA) following the manufacturer’s instructions. Then percentages of TNF or Granzyme B positive cells in CD3+ T cells or CD8+ T cells were labeled via intracellular staining and detected by flow cytometry. Antibodies for flow cytometry analysis were purchased from eBiosciences, USA. Matched isotype controls were used for each antibody to determine gates. FlowJo (Treestar, USA) software was used for the analysis of flow cytometry data. Standardized fluorescence intensities were calculated by dividing the median fluorescence intensities of specific antibodies by the median fluorescence intensities of isotype controls. The results are expressed as mean ± SD of three independent experiments.
In vivo mouse studies
C57BL/6 mice were obtained from Guangdong Medical Laboratory Animal Center, China, and kept in a specific pathogen-free (SPF) barrier facility at the Animal Center of Sun Yat-sen University Cancer Center. The female mice with 8–12 weeks old were used for all animal experiments. Experiments were approved by the institutional committee of Sun Yat-sen University Cancer Center, and conducted in accordance with protocols approved by the Guangdong Provincial Animal Care and Use Committee.
LLC cells (2 × 105 cells in 200 μL growth medium) were subcutaneously injected into the right flank of immunocompetent C57BL/6 mice. Tumor growth was measured with calipers every 3 days and the tumor volumes were calculated by applying the following formula: 1/2(length x width2). When tumors reached approximately 100 mm3, mice were randomized into control or experimental groups. A terminal event was defined as tumors reaching a size of 2000 mm3, at which point animals were euthanized [25, 26].
Mice were treated with niclosamide or rat anti-PD-L1 antibody (αPD-L1, clone 10F.9G2; BioLegend, USA) alone, the combination of niclosamide and αPD-L1, or saline and IgG2bκ (clone RTK4530; BioLegend, USA) by intraperitoneal injection (each group, n = 6–7). Niclosamide (20 mg/kg) or saline was administered intraperitoneally from day 13, every 5 days, after tumor implantation. Anti-PD-L1 antibody therapy (10 mg/kg) was administered intraperitoneally weekly on days 16, 23, 30, 37, and 44. Survival analysis was performed using Kaplan-Meier analysis and log-rank test.
Patients and tissue specimens
Tissue specimens were obtained from 28 patients with advanced NSCLC who received immunotherapy during the course of anti-cancer therapy at Sun Yat-sen University Cancer Center (Guangzhou, China). Clinical data was collected from pathology reports and unprocessed medical files. The study was conducted with the permission of the Ethics Committee of the Sun Yat-sen University Cancer Institutional Board, and all patients involved provided informed, written consent.
Histology and immunohistochemistry (IHC)
For IHC staining of the xenografts, tumor tissues were fixed, embedded, and sectioned (3 μm thick). Immunohistochemistry staining for human and mouse tissues was performed in accordance with standard procedures . The following antibodies were used: primary antibody CD3+ (dilution 1:200) or Granzyme B (dilution 1:400) (Cell Signaling Technology, USA) for mouse tissues, and anti-PD-L1 (dilution 1:1000) (E1L3N, Cell Signaling Technology) or anti-p-STAT3 (dilution 1:500) (D3A7, Cell Signaling Technology) for human tissues.
Since no human STAT3 crystal structure has been elucidated at present, mouse STAT3 (PDB ID: 4e68) was selected as receptor for docking as the identity of key residues and the high sequence identity (76.5%) sharing between human and mouse STAT3. Residues Lys591, Arg609, Ser636, Glu638 and residues Val637, Ile653 constitute the phosphotyrosine binding site and hydrophobic binding site of STAT3, respectively. Thus, we defined residues 591, 609, 636–638, 653 as putative binding sites to investigate the binding of niclosamide to STAT3. The docking simulations were carried out using Surflex module of SYBYL software (Tripos, Inc), which combines Hammerhead’s empirical scoring function with a molecular similarity method (morphological similarity) to generate putative poses of ligand . The crystal structure of STAT3 was retrieved from RCSB Brookhaven Protein Database (http://www.pdb.org/pdb/home/home.do) (PDB ID: 4e68). For molecular docking purpose, the substrate dsDNA M67 and crystal water molecules were removed, and all hydrogen atoms were subsequently added to the unoccupied valence of heavy atoms at the neutral state (pH 7). The small molecule niclosamide downloaded from pubchem database (https://pubchem.ncbi.nlm.nih.gov/) was employed to perform the docking process. Two parameters bloat and threshold, which determine how far a potential ligand should extend outside of the concavity and how deep into the protein the atomic probes, were used to define the protomol. For niclosamide, the protomol was generated using the residue approach, and the bloat and threshold were set to 0.4 and 1, respectively.
Statistical analysis was carried out using IBM SPSS Statistics 19 software or GraphPad Prism using Student’s t-test or one-way ANOVA or Dunnett’s test. All experiments were repeated in triplicate. Data are expressed as mean ± standard deviation (SD). Statistical significance was defined as P < 0.05.