3.1. Patients and specimens
All clinical samples were collected from the Department of Radiation Oncology, the First Affiliated Hospital of Nanjing Medical University. All subjects gave their informed consent before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee and Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University. A total of 88 OSCC patients were recruited in this study between 2017 and 2019. Clinical staging was based on the AJCC Cancer Staging Manual seventh edition(13). All patients underwent a 60 Gy total dose of involved-field radiation (2 Gy per fraction, 5 days per week) plus two cycles of concurrent chemotherapy of cisplatin and fluorouracil and two cycles of chemotherapy post-radiation. Radiation was delivered as described before(14). Blood samples were collected one day prior to definitive chemoradiation. Follow up studies included computed tomography, barium swallow, endoscopy and physical examination. All patients were followed up every 3 months until death or May 2020. In-field recurrence included primary lesion and involved regional lymph nodes inside PTV. Overall survival was defined as the interval between completion of radiation and death(14). We collected 30 ml whole blood from each patient using BD Vacutainer K2 EDTA Blood Collection Tubes (BD, USA), and we immediately centrifugated the whole blood at 4000 g for 10 min at 4 ℃ to get plasma. The plasma was then ultracentrifugated to collect EVs.
3.2. Cell culture and hypoxic treatment
Human OSCC cell lines (Te13, Te1 and Eca109) were obtained from American Type Culture Collection (ATCC, USA). All cell lines were cultured in RPMI-1640 medium (Gibco, USA) with 10% foetal bovine serum (FBS; Gibco, USA), 100 U/ ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 37 °C and 5% CO2 and were routinely examined for Mycoplasma contamination. To induce hypoxia (< 1% O2), cells were cultured at 37℃ in the same incubator in an AnaeroPack jar with AneroPack-Anaero (Mitsubishi, Japan) according to the manufacturer's instructions. The hypoxic environment was confirmed by hypoxia inducible factor 1 subunit alpha (HIF-1α) expression. Cells were irradiated by an RS 2000 Pro X-Ray Bio-irradiator (Radsource, USA).
3.3. EV isolation and identification
FBS was depleted of EVs by ultracentrifugation at 140,000 g and 4 °C for 16 hours, and the supernatant was collected and filtered using a 0.22 µm filter (Millipore, USA). EVs derived from blood samples and cell medium were isolated by differential centrifugation as previously described(15). Before EV isolation, cells were cultured in normal medium until 50% confluency and then washed with phosphate-buffered saline (PBS) three times; and the medium was replaced with RPMI-1640 with 10% EV-depleted FBS and cultured under normoxic or hypoxic conditions. After 48 hours, the cell culture medium was harvested (50 ml), and EVs were isolated by differential centrifugation as previously described. The EVs were used immediately for further experiments. The size distribution and concentration of EVs were analysed by nanoparticle tracking analysis (NTA) using a ZetaView particle tracker from ParticleMetrix (Meerbusch, Germany). We used a transmission electron microscope (TEM; JEM-1200EX, JEOL Ltd., Japan) to observe the structure of EVs. CD63, CD81 and Alix were used as exosomal markers, and calnexin was used as a negative control for EVs. PKH67 (Sigma-Aldrich, USA) was used to label EVs. Twenty-four hours after PKH67-labelled EVs were incubated with OSCC cells, DAPI was used for nuclei staining. The cells were visualized with a confocal fluorescence microscope (Leica, Germany).
3.4. Western blotting
Samples of cells and EVs were washed and resuspended in RIPA lysis buffer (Beyotime, Shanghai, China) with a protease and phosphatase inhibitor mixture (Millipore). Proteins were separated based on their molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk powder in Tris-buffered saline containing Tween 20 (TBST) for 2 hours, and then the membranes were incubated at 4 °C overnight with specific primary antibodies (details are listed in Table S1). The membranes were rinsed in TBST for three times (10 minutes each time) and were incubated in secondary antibodies at room temperature for 2 hours and then were washed again in TBST (three times,10 minutes each time). Protein expression levels were detected by ECL Plus (Millipore) using a Bio-Imaging System (Bio-Rad, USA).
3.5. Quantitative real-time PCR (qRT-PCR)
Following the manufacturer's instructions, total RNA was extracted with TRIzol reagent (Invitrogen, USA). For qRT-PCR of mRNA, total RNA was further reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Japan). For miRNA RT-PCR, we used the Mir-X miRNA First-Strand Synthesis Kit (Takara) to perform reverse transcription. The reactions were processed using a Real-time PCR System (Applied Biosystems 7500, USA) with a TB Green Premix Ex Taq Kit (Takara). The primers of target mRNA/miRNA were obtained from Wcgene Biotech (Shanghai, China). For normoxic cell samples, expression levels of genes and miRNAs were normalized to GAPDH or U6, respectively. Since hypoxia has an influence on the GAPDH expression, β-actin was obtained to evaluate gene levels in hypoxic cells(16). For EV samples, expression levels were normalized to external controls cel-miR-39 (RiboBio, Guangzhou, China). The results are shown in the form of relative expression calculated by the 2−ΔΔCT method.
3.6. Vector construction, lentivirus production and cell transfections
LV2-hsa-miR-340-5p-mimic vector and LV2-hsa-miR-340-5p-inhibitor vector were constructed as lentiviral vectors (GenePharma, Shanghai, China). Cells were infected when grown to 40–50% confluence and selected with puromycin for stable transfection. pcDNA3.1 vector containing ETS1, KLF10 and the negative control were purchased from GenePharma. siRNAs and mimics were purchased from RiboBio. Lipofectamine 3000 reagent (Invitrogen) was used for transfection. Sequences of vector were listed in Table S1.
3.7. 5-Ethynyl-2ʹ-deoxyuridine (EdU) assay
We measured cell proliferation using an EdU assay kit (RiboBio). Twenty-four hours after coculture with EVs, cells were digested and seeded into 24-well plates (2 × 104 cells/well) and cultured with RPMI- 1640 (10% FBS) for 24 hours before adding EdU (50 µmol/L). According to the protocols, cells were then incubated for 2 hours at 37 °C, fixed in 4% paraformaldehyde for 30 minutes and permeabilized with 0.5% TritonX-100 for 10 minutes at room temperature. After washing with PBS, 1 × ApolloR reaction cocktail was added to react with the EdU for 30 minutes. Subsequently, Hoechst33342 was added for 30 minutes to visualize the nuclei. Images of cells were obtained under a Nikon microscope (Nikon, Japan). Proliferation was assessed using the mean number cells in three fields for each sample.
3.8. Luciferase report assay
Sequences corresponding to the 3ʹ-UTR of KLF10 mRNA and containing the wild-type or mutated miR-340-5p binding sequence were synthesized by GeneScript (Nanjing, China). We inserted these sequences into the XbaI and SacI sites of the pmirGLO dual-luciferase miRNA target expression vector (Promega, USA). They were cotransfected with vectors and miR-340-5p-mimic/miR-340-5p-inhibitor or miR-NC by using a Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Luciferase activity was measured by Luciferase Reporter Assay System (Promega).
3.9. RNA immunoprecipitation (RIP)
The EZ-Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore,) was used to conduct the RIP assay. Cells were collected and lysed by pre-cooled lysis buffer supplemented with 1 mM PMSF, protease inhibitor and RNase inhibitor. Cells were incubated with RIP buffer containing magnetic beads conjugated with human anti-Argonaute 2 (Ago2) antibody (Millipore) or normal IgG (Millipore). Precipitate was digested and then co-immunoprecipitated RNA was isolated for PCR.
3.10. Colony formation assay
We seeded 200, 400, 800, 2000 and 5000 cells/well in 6-well plates one day before exposure to 0, 2, 4, 6 and 8 Gy irradiation, respectively. After two weeks, each well was washed with PBS for three times, then fixed with 4% paraformaldehyde for 30 minutes and finally stained with crystal violet. Colonies were counted with the naked eye. The survival curve was calculated using a single-hit, multi-target model.
3.11. Cell apoptosis analysis
Cell apoptosis was carried out using an apoptosis detection kit (Vanzyme, Nanjing, China) following the manufacturer's instructions. At 72 hours post-irradiation with 8 Gy, cells were digested and then resuspended in 500 µl of binding buffer with 5 µl of annexin V-FITC solution and 5 µl of propidium iodide (PI), and the cells were stained at room temperature for 15 minutes in the dark. Flow cytometry (FACScan; BD Biosciences, USA) and FlowJo software (BD, USA) were used to analyse the cells.
3.12. Immunofluorescence assay
A total of 5 × 104 cells were seeded into a confocal laser dish one day before 8 Gy irradiation. Four hours post-irradiation, cells were fixed in 4% paraformaldehyde at room temperature for 30 minutes and permeabilized in 0.1% Triton X-100 for 2 hours. Cells were then blocked with 5% BSA for 90 minutes and washed with PBS. After incubation with the primary antibody γ-H2AX (1:400; Cell Signalling Technology) overnight at 4℃, the cells were washed with PBS and incubated with an Alexa Fluor 555-conjugated secondary antibody (Beyotime) for 90 minutes. Cells were washed with PBS and treated with DAPI staining solution for 20 minutes and then observed using a confocal fluorescence microscope (Leica).
3.13. Tumour xenograft in nude mice model
This study was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-1901012). BALB/c nude mice were purchased from the Animal Center of Nanjing Medical University. A total of 2 × 106 cells were injected subcutaneously into the flanks of nude mice. Tumours were measured by callipers every 5 days using the following formula: volume = (width2 × length)/2. When the tumour volume reached 50 mm3, 10 µg (50 µl) EVs or 50 µl PBS were injected into tumour once daily for 5 days. Meanwhile, tumours were irradiated by an RS 2000 Pro X-Ray Bio-irradiator (Radsource, USA) with 2 Gy per day for 4 consecutive days starting from the second day of EV injection. Two weeks post-IR, the mice were euthanized. In addition, we injected NC-Te13 cells and miR-340-5p mimics Te13 cells into the nude mouse flanks to study the effect of miR-340-5p. When the tumour volume reached 50 mm3, four fractions of radiation (2 Gy/f) were applied to the tumours, and the mice were sacrificed 14 days after the radiation was completed. Lead shields were used to prevent radiation injury.
3.14. Immunohistochemistry
Immunohistochemistry was done as previously reported(17). Staining intensity was scored according to 4 grades: 0 (no staining), 1 (weak staining), 2 (intermediate staining), or 3 (strong staining). The staining percentages were divided into 4 grades: 0 (no positive), 1 (༜25% positive), 2 (25%-50% positive) and 3 (༞50% positive). The product of staining intensity and percentage was used as the final staining score.
3.15. TUNEL assay
The TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche, Germany). Following the manufacturer’s protocols, sections were deparaffinized and rehydrated. Antigen retrieval was performed using hot 0.1 M citrate buffer pH 6.0, after which the sections were incubated with the TUNEL reaction mixture (containing TdT and fluorescein-conjugated dUTP) for 1 hour at 37℃. DAPI was used to stain nuclei. The apoptotic cells were analysed using a fluorescence microscope (Leica). For the negative control, TdT was not included in the reaction mixture.
3.16. Statistical analysis
All experiments in this study were carried out in triplicates. Differences between groups were determined using Student's t test, one-way ANOVA and two-way ANOVA. A chi-squared test was used to detect differences in clinical data. The patients were divided into high expression and low expression groups based on the median of gene expression. Kaplan-Meier analysis was used to compare recurrence in OSCC patients. Receiver operating characteristics (ROC) curves were constructed to evaluate the accuracy of prediction. STATA 14.0, SPSS 22.0 and GraphPad Prism 8.0 software were used to perform statistical analysis, and a p value < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).