Human tissue samples
Tissue samples were obtained from 180 confirmed NSCLC tumors and 30 corresponding metastatic lymph nodes resected between December 2013 and September 2015 and assessed in The Department of Pathology, Affiliated Tongji Hospital of Huazhong University of Science and Technology Tongji Medical College (Wuhan, China). The samples were obtained from 127 males and 53 females (mean age, 50.3 ± 4.7 years; range, 21 to 77 years). Clinicopathological parameters, including age, sex, smoking index, pathological diagnosis and clinical stage, were obtained from the Tongji hospital records. The histological characterization and clinical–pathological staging of the samples was determined according to World Health Organization criteria[13]. Paraffin-embedded tumor specimens were used to create tissue microarray (Outdo Biotech Co., Ltd., Shanghai, China) blocks with 2-mm diameter cores for immunohistochemical (IHC) staining. Two tissue cores were obtained from each patient. The baseline characteristics of the patients are shown in Table 1. Informed consent was obtained before surgery. All patients involved consented to participate under the Clinical Patient ethical statement. Study approval for the study was obtained from the Research Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IRB ID number 20141101).
Details of the 30 metastatic lymph node samples with IIA-IIIB NSCLC and lymph node metastasis were provided in our previous study[13]. Patients with lymphadenitis or primary malignancies of the lymph node were excluded. Palliative care or surgical biopsy following informed consent was administered to 32 patients with inoperable Stage IIIb-IV primary NSCLC.
Immunohistochemistry
Immunohistochemical staining was carried out using the avidin–biotin peroxidase method [13, 15]. All sections were deparaffinized in xylene, rehydrated in alcohol, incubated in hydrogen peroxide, blocked in 10% goat serum and then incubated overnight in 0.3% H2O2 at room temperature with primary antibodies (rabbit anti-human TLR4 polyclonal antibodies, 1:100, Abcam, Cat: ab13556 and rabbit anti-human ERβ monoclonal antibodies, 1:100, Abcam, Cat: ab3577), Immunoreactivity scores of the cancer tissue samples were determined based on the staining intensity and positive staining area according to the method described in Tang et al.[14]. A staining index (values 0–16), obtained as the intensity of positive staining (negative(1 scores), weak(2 scores), moderate(3 scores), or strong(4 scores)) and the proportion of immune-staining positive cells of interest (< 25%(1 scores), 25% to 50%(2 scores), 50% to 75%(3 scores), ≥75%(4 scores)) were calculated. A score of 1-16 was obtained by multiplying the staining intensity and positive cell proportion. A total score > 12 was defined as high expression, a score ≤ 8 was defined as low expression, and a score ≤ 4 was defined as negative expression.
Cell culture and associated reagents
The human NSCLC cell lines A549 and H1793 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The culture condition was described in previous study[13] and were maintained in RPMI 1640(A549) or DMEM/F-12(H1793) supplemented with 10% fetal bovine serum (FBS). The NSCLC cells were treated with E2 (Sigma-Aldrich, St. Louis, MO, USA), fulvestrant, also known as Faslodex or ICI-182780 (7α-[9-[(4,4,5,5,5-pentafluoropentyl)-sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17β-diol, Cayman Chemical), and CLI-095, also known as TAK242 (ethyl(6R)-6-[N-(2-chloro-4- fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate, InvivoGen, USA) either alone or in combination. The doses of these drugs used in vitro and in vivo were comparable to previously described doses[13, 15, 27]. Each group of cells was treated for 48 h and harvested for further analysis. Cell culture experiments were performed using reagents formulated in 100% DMSO.
Western blot analyses and Immunofluorescence
Western blot analyses and Immunofluorescence were performed as described by us previously[13, 27]. Briefly, total protein extract for each cell line was dissolved in lysis buffer and equal amounts of protein (40 μg) were analyzed by immunoblotting. The Immunofluorescence coverslips were observed under a fluorescence microscope (Olympus, Tokyo, Japan).
The primary antibodies used for the Western blot analyses and Immunofluorescence included rabbit anti-human ERβ (1:1,000) from Abcam (Cat: ab3577), rabbit anti-human TLR4 (1:1,000) from Abcam (Cat: ab13556), rabbit anti-human myd88 (1:1,000) from Proteintech(Cat: 23230-1-AP), and mouse anti-human GAPDH (1:10,000) from Cell Signaling Technology (CST; Cat: 51332; USA).
Wound-healing assay
NSCLC cells were seeded in six-well plates for 24 h. After growth to confluence, the surface of the plate was scraped with a 200 μL pipette tip to generate a cell-free zone, and then incubated with RMPI 1640 containing 10% FBS for 24 h. Cells were photographed using a phase-contrast microscope (100×) as previously described[13].
In vitro Transwell® migration and invasion assays
Transwell migration and invasion assays were performed as described by us previously[13, 27]. Transwell® Permeable Supports (24-well supports, inserts were 6.5 mm in diameter) (Corning, NY, USA) were used. In brief, NSCLC cells suspended in serum-free medium were added to the upper chamber at various densities depending on the cell line. Invasion assays were evaluated based on the number of cells invading a Transwell® membrane coated with Matrigel® (BD Biosciences, Bedford, MA, USA), and counting was conducted using an Olympus microscope (Olympus, Tokyo, Japan) at 100× magnification. Four fields were randomly selected for analysis. Detailed procedures are described elsewhere.
Migration assays were performed in 24‑well Transwell® chambers containing polycarbonate filters with 8‑μm pores without Matrigel®. The remaining steps were identical to those described for the Transwell® invasion assays.
3D spheroid invasion assay
The Cultrex® 3D Spheroid Cell Invasion Assay Kit (Catalog: 3500-096-K, Trevigen, Gaithersburg, MD) was utilized for this procedure. A total of 1 ×105 cells in 500 mL of medium containing 2.5% Matrigel and 5 ng/mL Spheroid Formation ECM were plated in 24-well plates coated with a collagen/Matrigel mixture. Spheres with protrusions were considered positive for cell invasion. Distances between the invasive cell frontier and spheroid edge were measured at 0, 72 and 144 h using an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan). Each experiment was repeated twice, and each procedure was performed in triplicate.
Fluorescent gelatin degradation assay
A QCMTM Gelatin Invadopodia Assay kit (Green) (Catalog: No.ECM670, Millipore, USA) was utilized for this procedure. Coverslips were cleaned with 20% nitric acid and coated with poly-L-lysine in a 24-well plate. Poly-L-lysine was fixed with 0.5% glutaraldehyde before adding FITC-conjugated gelatin. A thin layer of FITC-conjugated gelatin was applied to the coverslips and crosslinked using glutaraldehyde on ice for 10 min. Crosslinking was continued at room temperature for an additional 30 min. The coverslips were rinsed with PBS, incubated with 5 mg/mL sodium borohydride at room temperature for 3 min, rinsed again with PBS, incubated with 70% EtOH for 10 min, and dried at 37°C for 15 min in a CO2 incubator. One hour before plating cells, the coverslips were quenched with RPMI-1640 medium containing 10% FBS at 37°C. Cells were plated on the FITC-conjugated gelatin-coated coverslips and cultured in RPMI-1640 medium for 24 h to quantify the formation of invadopodia. Images were visualized by confocal microscopy (Olympus, Tokyo, Japan).
Statistical analysis
Statistical analysis was performed using SPSS 19.0 statistical software (IBM, New York City, USA). The χ2 test was performed to analyze the correlations among ERβ and TLR4 expression and clinicopathological parameters, and Spearman’s rank correlation was used to analyze the correlations between ERβ and TLR4 expression in primary NSCLC and metastatic lymph node tissue samples. Comparisons between groups were analyzed using an unpaired t-test. Data are presented as the mean ± SE. The remaining data are presented as the mean ± SD. P values < 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism (version 7.00, GraphPad, San Diego, CA, USA).