2.1 Cell culture and regents
The human NPC cell lines, 5-8F, 6-10B, and HK-1 were donated by the Sun Yat-sen University Cancer Center. Cells were cultured in 1640 medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Thermo Scientific). Human CAFs and normal fibroblasts (NFs) were extracted from fresh NPC tissues and matched to normal nasopharynx tissues from patients newly diagnosed with NPC. Fibroblast isolation was performed as previously described. Briefly, fresh tissues were cut into pieces of 2 to 3mm and cultured in high-glucose DMEM medium (Gibco) for approximately one week until fibroblasts appeared. Specimens were obtained with written informed consent from patients with NPC enrolled at Nanfang Hospital, Southern Medical University. In functional studies, NFs and CAFs were used at than ten passages. All cells were cultured in a humidified incubator containing 5% CO2 at 37°C.
Human recombinant IL-8, HGF, and TNF-α were purchased from Peprotech (Suzhou, China). An antagonist of IL-8 receptor (Repertaxin), inhibitor of NF-κB activation (caffeic acid phenethyl ester [CAPE]), and a lactate inhibitor (sodium dichloroacetate, DCA) were purchased from Selleck Chemicals (Houston, USA); Tranilast was purchased from Target Mol (Massachusetts, USA).
2.2 Establishment of radiation-resistant (IRR) 5-8F IRR cells
Establishment of IRR cell lines was performed as previously described. In brief, cells were treated twice with irradiation at a graded dose of 2, 4, 6, 8 and 10 Gy, respectively. A total IR dose of 60 Gy was administered with the entire selection procedure. The final surviving 5-8F cells were verified, defined as radioresistant 5-8F cells, and termed 5-8F IRR. The parental 5-8F cells were treated without irradiation. Cells within 5 - 10 passages were used for experiments once the 5-8F-IRR cells were successfully established.
2.3 Cell viability assay
CAFs were subjected to a cell viability assay using Cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer’s instructions. In brief, CAFs were cultured in medium containing Tranilast at a concentration of 200 μM and 400 μM, respectively for two days and subsequently cultured at the density of 1.5 × 103 cells per well in triplicate in 96-well plates containing Tranilast-free medium. After culturing for 8 h, CAFs were applied to CCK-8 assay and a subsequent detection was performed at the indicated time points. To detect the impact of Repertaxin and CAPE on NPC cell line proliferation, cells were cultured in medium containing a wide-range of reagent concentrations (0.01, 0.1, 1, 10, and 100 μM for CAPE and 1, 10, 40 μM for Repertaxin) for 2 - 4 days and then subjected to a CCK-8 assay.
2.4 Colony forming assay
A total of 5-8F, 6-10B, and HK-1 cells were cultured in six-well plates for 12 h at various densities according to the aim of each experiment. The cell density was listed as follows: 300 (0 Gy), 400 (2 Gy), 1000 (4 Gy), 2000 (6 Gy), 4000 (8 Gy) per well for 5-8F and 6-10B and 500 (2 Gy), 10000 (8 Gy) per well for HK-1. After a 2-week incubation, the six-well plates were obtained and colonies with more than 50 cells were counted. The surviving fraction (SF) was estimated using the following formula: SF = (number of colonies formed/number of cells seeded × plating efficiency of the control group), where plating efficiency was calculated as the ratio between colonies observed and the number of cells plated. Dose-response colony survival curves were plotted accordingly.
2.5 Cytokine assay
CAF and NF cells were cultured in serum-free medium when grown to 80% confluence. After incubating for two days, the medium was collected and concentrated before dialysis. After labeling with biotin, the samples were applied to an antibody array chip (R&D; Cat ARY022B, LabEx) overnight. The following day, the chips were reacted with streptavidin-conjugated fluorescence dye, and detected with a chemiluminescence imager (Chemi Scope 6300). Data were normalized to the total protein.
2.6 RNA sequencing and tissue microarray
Total RNA of parent 5-8F and 5-8F IRR cells were extracted using TRIzol reagent (Invitrogen Life Technologies, CA) according to the manufacturer’s instructions. The prepared RNA was then subjected to sequencing on a BGISEQ-500 platform at Beijing Genomics Institution (www.genomics.org.cn, BGI, Shenzhen, China). The level of gene expression was quantified and the NOISeq method was performed to screen for differentially expressed genes (DEGs) as previously described. A tissue microarray was conducted by Shanghai Biotechnology Corporation using Affymetrix Genechip according to manufacturer’s protocol.
2.7 Radiosensitivity index (RSI) analysis and gene set enrichment analysis
An RSI index served as an indicator for radiosensitivity, and was calculated as previously described. In brief, head and neck squamous carcinoma (HNSCC) patients in the TCGA database and NPC patients in the GSE12542 dataset were divided into two groups based on the median of CAFs score using an online tool (https://gfellerlab.shinyapps.io/EPIC_1-1/). Gene Set Enrichment Analysis (GSEA) software version 3.0 (Broad Institute, USA) was used to analyze GSE48501 and a human microarray containing radioresistant and radiosensitive NPC samples. A threshold of P ≤ 0.05 was applied for the analysis. Data for GSE48501 and GSE12542 were downloaded from the NCBI Gene Expression omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/).
2.8 Lactate detection
Lactate was detected using a lactic acid measurement kit (Junji Biotechnology Co, China) according to the manufacturer’s protocol. Briefly, blank medium and conditioned medium produced by NFs and CAFs were prepared and subjected to a chromogenic reaction with the indicated reagents. The absorbance value was detected by a microplate reader machine (BIO-RAD 689). Data were normalized to the lactate standards.
2.9 Comet assay
A comet assay was performed using a DNA Damage Detection Kit (Keygen, China). The cells were irradiated as described at dose of 2 Gy and treated in accordance with the experimental design. The following day, cells were harvested and suspended in PBS containing 1% low-melting agarose and layered onto adhesive microscope slides previously covered with 0.5% normal-melting agarose. The cells were dipped in a specific lysed buffer at 4°C for 2 h. Next, the DNA was uncoiled and unwound in an alkalescent electrophoresis buffer for 30 min. Electrophoresis was performed and the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution for 10 min in a dark room. The slides were examined with an Eclipse fluorescence microscope (Nikon, Japan).
2.10 Real-time quantitative PCR
Total RNA was extracted from CAFs using TRIzol reagent (Invitrogen Life Technologies, CA) as instructed by the manufacturer’s protocol. SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, MA) was used for reverse transcription according to the manufacturer’s protocol. Real-time quantitative PCR (qRT-PCR) was implemented to measure specific mRNA expression using an ABI7500 FAST system with TaqMan Reverse Transcription Reagents and SYBR Green PCR MasterMix (Applied Biosystems, CA, USA). GAPDH was used as a loading control. The specific primers were listed as follows: GAPDH, forward: 5′ -GGAGCGAGATCCCTCCAAAAT-3′ and reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′. IL-8, forward: 5′-GTGCAGTTTTGCCAAGGAGT-3′ and reverse: 5′-CTCTGCACCCAGTTTTCCTT-3′. HGF: forward: 5′-TGGGCCATTCTATTCCCCC-3′ and reverse: 5′-CATGGGGTCAAGCTTCCAGT-3′. The △Ct expression values for amplification were calculated by normalizing to an internal control.
2.11 Conditioned medium derived from human NFs and CAFs
CAFs and matched non-malignant NFs were cultured in 25 mL culture flask with high-glucose DMEM (Gibco) supplemented with 10% FBS at same density overnight, respectively. The cells were refreshed with serum-free DMEM and the supernatants were harvested after 48 h. Next, the cell pellet was removed after centrifugation and the conditioned medium (CM) was acquired and stored at -80°C for future experimentation.
2.12 Western blotting analysis
Cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with phosphatase and a protease inhibitor cocktail (Roche). The proteins were subjected to 10% SDS-PAGE followed by transfer to PVDF membrane and subsequent incubation with specific primary antibodies at 4 °C overnight. Membranes were then incubated with respective secondary antibodies (FuDe, China) for 1 h at room temperature. The primary antibodies included mouse monoclonal anti-GAPDH (60004-1-IG, Proteintech, USA), rabbit monoclonal anti-α-SMA (ab124964, Abcam, USA), rabbit monoclonal p65 (8242, CST, USA), rabbit monoclonal p-p65 (3039, CST, USA), mouse monoclonal IKB-α (4841, CST, USA), and rabbit monoclonal γ-H2AX (9718, CST, USA). GAPDH was used as an internal reference.
CAFs were seeded into a confocal dish and incubated overnight. The cells were washed three times with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min. Next, 0.5% Triton X-100 was used for permeabilization and the samples were blocked with goat serum albumin. Subsequently, the samples were incubated with primary antibodies targeted to α-SMA (1:400) at 4°C overnight, followed by an incubation with an Alexa fluor-488-conjugated secondary antibody. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI), and CAFs were observed with an Eclipse fluorescence microscope (Nikon, Japan).
2.14 IL-8 RNA knockdown in CAFs
A knockdown of IL-8 in CAFs was performed using small interfering RNA (siRNA) (RIBOBIO, China) with Lipofectamine 2000 (Invitrogen, CA) for 10 h of transfection. The specific sequence targeting IL-8 was listed as follows: GCCAAGGAGTGCTAAAGAA.
2.15 Patients and clinical samples
All patients were treated with intensity-modulated radiation therapy with a radical dose ranging from 60 Gy to 70 Gy. Radioresistance was defined as the local relapse or recurrence of the ever irradiated area after 6 months of radiotherapy via enhanced computed tomography or enhanced magnetic resonance imaging. Human NPC samples were obtained from Nanfang Hospital, Southern Medical University, including 6 cases of radioresistant tissues and 16 cases of radiosensitive tissues. The collection of clinical samples for research was approved by the Ethics Committee of the Nanfang Hospital
2.16 Immunohistochemistry assay
Each of the staining processes were conducted according to the manufacturer’s protocol. Primary antibodies consisted of rabbit monoclonal anti-α-SMA (ab124964, Abcam, USA) at a dilution of 1:400. The level of α-SMA expression was evaluated based on the staining intensity and percentage of positively-stained CAFs. Final scores were assessed as the total evaluation of staining intensity (0 for negative, 1 for weak, 2 for moderate and 3 strong) plus percent positivity (0 for 0%, 1 for 1% - 25%, 2 for 26% - 50%, 3 for > 50). The IHC evaluation was performed by two independent pathologists.
2.17 Mouse irradiation assays
Three-week-old male BALB/c nude mice were purchased from Southern Medical University Laboratory Animal Center (Guangzhou, China). Experiments involving animals were approved by the Institutional Animal Care and Use Committee of the Southern Medical University. Mouse models were generated by a subcutaneous inoculation of 5-8F cells alone or combined with human cancer-associated fibroblasts at a 1:1 ratio. The number of cancer cells in each injection was 1 × 106 for one flank side. Mice were divided into three groups and received irradiation treatment at a 8Gy × 3 schedule 9 days after injection. Before irradiation, one co-injection group was subjected to an orthotopic injection of 200 μM Tranilast in 100 μL daily for three days. The tumor volume was determined at the indicated time points with the following formula: tumor volume (mm3) = ½ × longest diameter 2 × shortest diameter. At 3 weeks post-irradiation, the mice were sacrificed and the tumors were excised.
2.18 Statistical analysis
Statistical analysis of the data was performed using SPSS software version 20.0. Both paired and unpaired t-tests were performed to analyze the data between two experimental groups. Mann-Whitney tests were used to calculate the P values for IHC staining quantification. Data were presented as the means ± standard deviation (SD). Statistical significance was defined as a P-value less than 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance.