Four cases of fresh OSCC tissues were obtained from the First and Second Affiliated Hospitals, Dalian Medical University. CAFs were isolated from OSCC tissues and named CAF-S1, CAF-S2, CAF-S3 and CAF-S4, respectively . Normal fibroblasts (NFs) were isolated from normal gingival tissues of a healthy adult during tooth extraction. CAFs and NFs were cultured in DMEM/F12 medium (Gibco, Grand Island, NY, USA). UM-SCC6 was a kind gift from Peking Union Medical University and cultured in DMEM/High Glucose medium (Hyclone, Logan, UT, USA). All cells were supplemented with 10% fetal bovine serum (FBS, ScienCell, Carlsbad, CA, USA), 100U/mL penicillin and 100U/mL streptomycin (Hyclone) at 37°C in a humidified 5% CO2 incubator.
sEV separation and labeling
Bovine sEVs were depleted from FBS by ultracentrifugation. When cells reached 80% confluency, fresh DMEM/F12 medium with 2% sEV-depleted FBS was used to culture cells for another 72 h as conditioned medium (CM). Then, CM was collected and differentially centrifuged 500 g for 10 min, 2,500 g for 20 min, 12,000 g for 30 min and 100,000 g for 70 min. Then, the pellet was diluted in 20 mL phosphate buffer saline (PBS) and ultracentrifuged at 100,000 g for 70 min. For plasma EV separation, blood was collected into an EDTA-K2 anticoagulant tube, mixed immediately to avoid clotting, and sequentially centrifuged at 1,500 g and 2,400 g for 10 min at 4 °C. The supernatant was collected and diluted at ratio 1:1 with PBS followed by ultracentrifugation.
The protein concentration of sEVs was measured by BCA kit (Beyotime Biotechnology, shanghai, China). sEVs were labeled with PKH67 (Sigma-Aldrich, Louis, MO, USA) according to the company’s instruction. Labeled sEVs were re-separated by ultracentrifugation at 100,000 g for 70 min.
Transmission Electron microscopy (TEM)
sEVs in PBS were placed on a formvar carbon-coated grid for 20 min. The sample was negatively stained with 1% phosphotungstic acid solution for 5 min. For immunogold labeling, purified sEVs in PBS were placed on formvar carbon-coated grid for 20 min, washed with PBS twice and incubated 50 mM glycine to quench free aldehyde groups. After the grids were washed, they were
blocked using PBS containing 5% bovine serum albumin (BSA) for 1 h, and incubated with primary antibodies against CD63 (10 µg/mL, Abcam, Cambridgeshire, UK), CD9 (10 µg/mL, Abcam), LOX (10 µg/mL, Abcam) overnight at 4℃. Then, the samples were incubated with goat anti-rabbit IgG conjugated to 5 nm-colloidal gold particles (Invitrogen). Then the grid was washed with PBS contained 0.1%BSA and fixed with 2.5% glutaraldehyde for 15 min then thoroughly washed with deionized water. Samples were counterstained with phosphato-tungstic acid for 5 min. Images were taken using a transmission electron microscope (JEM-2000EX*, Japan Electronics, Japan).
Western Blot analysis
Cells or sEVs were lysed by RIPA buffer (Millipore Corporation, Billerica, MA, USA) supplemented with PMSF (100 mM, Solarbio, Beijing, China) and cocktail protease inhibitors (Sigma-Aldrich) at 4℃ for 15 min. Protein concentration was determined by BCA kit (Beyotime Biotechnology). Equal amount of proteins was separated by sodium dodecyl sulfate-polyacrylamide gel. Proteins on gel were transferred onto a nitrocellulose membrane (Millipore Corporation). Then, the membrane was blocked with 5% fat-free milk for 2 h followed by primary antibodies incubation at 4℃ overnight. Primary antibodies included CD63 (1:500; Abcam), CD9 (1:500; Abcam), HSP70 (1:500; Abcam), CALNEXIN (1:500; Proteintech, Wuhan, China), LOX (1:600; Abcam), POSTN (1:500; Abcam), BMP-1 (1:300; Bioss, Beijing, China), FN (1:500; Abcam), integrin α2 (1:500, Abcam), integrin α4 (1:500; Abcam), integrin β1 (1:500; Abcam), vimentin (1:1000; Abcam), FSP-1(1:1000; Abcam), FAP (1:500; Abcam), α-SMA (1:1000; Proteintech, Wuhan, China), and GAPDH (1:3000; Proteintech). After the membrane incubation with horseradish peroxidase (HRP)-labelled IgG (H+L) as secondary antibody (1:3000; Proteintech) for 1 h. Protein bands were detected with Enhanced chemiluminescence detection system (ChemiDoc XRS, Bio-Rad).
RNA isolation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted using Trizol Reagent (Invitrogen). The PrimerScript RT reagent Kit (TaKaRa, Dalian, China) was used for reverse transcription. Quantitative real-time PCR reaction was performed with SYBR Premix Ex Taq reagent kit (TaKaRa) using Thermal Cycler Dice Real Time System (TaKaRa). GAPDH was used as an internal control. The primers for POSTN and GAPDH were purchased from TaKaRa. Primer sequences were as following: POSTN (forward): 5′-CCA TCA CAT CGG ACA TAT TGG A-3′, (reverse): 5′-TGC TCC TCC CAT AAT AGA CTC A-3′; GAPDH (forward): 5′-GTG AAG GTC GGA GTC AAC G-3′, (reverse): 5′-TGA GGT CAA TGA AGG GGT C-3′. Each experiment was repeated at least three times.
Protein A/G plus-magnetic beads (SelleckChem, Shanghai, China) was incubated with anti-POSTN antibody (Santa Cruz Biotechnology) under rotation for 15 min, followed by incubating with the protein extract from cells or sEVs for 15 min. The immunoprecipitated proteins were washed and resuspended in loading buffer, denatured and submitted to western blot analysis.
Enzyme-linked immunosorbent assay (ELISA)
To detect LOX or POSTN in sEVs, ELISA kits was purchased (LOX: Elabscience #H0174c; POSTN: Elabscience #H2452c). sEVs separated from CM were prepared for the serial dilution were added into each well (100 µL/well). Plasmal sEVs (100 µg) or sEV-excluded plasma (100 µg) were added into each well (100 µL/well). After 90 min, biotinylated LOX or POSTN antibody was added into each well and incubated at 37℃ for 1h. Then, horseradish peroxidase-conjugated streptavidin (100 µL) was added and incubated at 37℃ for 30 min followed by incubation with 3,3',5,5'-tetramethylbenzidine substrate reagent at 37℃ for 15 min. After adding 50 µL of stop solution to each well, the absorbance at 450 nm was measured with a microplate reader (ThermoFisher scientific, Waltham, MA, USA). To block the sEV surface LOX or POSTN, LOX or POSTN blocking antibodies (10 µg/mL) in 100 µL PBS were used to incubate with sEVs at 4℃ overnight, washed with PBS (20 mL), and pelleted by ultracentrifugation to remove non-bound antibodies. Each experiment was repeated at least three times.
Two POSTN gene-specific short interfering RNA (siPOSTN-1: 5′ - CCC AUG GAG AGC CAA UUA UTT-3′ and siPOSTN-2: 5′-CUC UGA CAU CAU GAC AAC AAA UTT-3′) and a negative control siRNA (si-NC: 5′-UUC UCC GAA CGU GUC ACG UTT-3′) were synthesized by Genepharma (Shanghai, China). They were transfected into cells with Lipofectamine™ 2000 reagent (Invitrogen) according to manufacturer’s instructions. After 48 h, total RNA and protein were isolated from transfected cells. sEVs were separated from the CM of transfected cells.
sEV treatment of human NFs
To assess the interactions of CAF sEVs with stromal cells and ECM, NFs (1×105 cells/well) labeled with CellTracker Red CMTPX Dye (Invitrogen) were cultured in a 12-well plate for 12 h or 72 h. Then, PKH67-labeled sEVs (20 μg/well) were added into each well and incubated for 12 h. After washing with PBS, NFs were fixed with 4% paraformaldehyde for 15 min. Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Images were recorded by an inverted microscope (Olympus IX71, Tokyo, Japan).
To test collagen cross-linking, NFs (1×105 cells/well) were seeded in a 6-well plate and cultured for 72 h. Purified sEVs (100 µg/well) were added into each well and incubated for 12 h. Fox LOX inhibition, sEVs (100 µg) were pre-incubated with β-amino propionitrile (BAPN, 200 µM) and the LOX-blocking antibody (10 µg/mL) at 4℃ overnight. The immature crosslinks, DHLNL and HLNL, and a mature crosslink, PYD, were quantified by ELISA assay. ELISA kits (DHLNL: Jonln #JL47954, HLNL: Jonln #JL47940, PYD: Jonln #JL19698) were purchased from Shanghai JONLN Reagent Co., Ltd., China. The absorbance at 450 nm was measured using a microplate reader (ThermoFisher scientific).
sEV treatment of human collagen matrix
To confirm sEVs bond to collagen matrix, type I collagen (3 mg/mL, Corning, Bedford, MA, USA) was mixed with Matrigel (Corning) at ratio 2:1 and added to a 48-well plate and incubated for 30 min at 37℃. Then PKH67-labeled sEVs (20 µg/well) were added into the collagen I-containing wells and incubated at 37℃ for 12 h. To inhibit integrin α2β1-mediated collagen binding, sEVs were pre-incubated with 2 µM TCI-15 (TOCRIS, Oxfordshire, UK) at 37℃ for 1 h. After washing with PBS, images were recorded by an inverted fluorescent microscope (Olympus IX71).
To visualize sEV-stimulated collagen crosslinking, collagen I (5 µg) was mixed with 20 µg sEVs and incubated for 12 h at 4℃. PBS was used as a control. The samples were placed on a formvar carbon-coated grid for 20 min, then negatively stained with 1% phosphotungstic acid solution for 5 min. Any excess of stain was wicked away and grid was air-dried at room temperature. Images were recorded using a transmission electron microscope (JEM-2000EX*).
To detect collagen crosslinks, sEVs (100 µg) mixed with collagen I (Corning) were added into a 24-well plate and incubated for 12 h. PYD, DHLNL, and HLNL were quantified by ELISA assay.
Tumor spheroid assay in vitro
Collagen I (2 mg/mL) was mixed with Matrigel in the ratio 1: 2 by volume. A 96-well plate was pre-coated with the collagen I-Matrigel mixture (30 µL/well). UM-SCC6 cells were suspended in the collagen I-Matrigel mixture and added to the precoated 96-well plate (3.75×104 cells in 75 µL/well). After the mixture gelled, DMEM/High Glucose medium (100 µL/well) was added into each well. After 2 days, tumor cells formed spheroids in the wells and CAF sEVs (100 µg) were added into each well with or without BAPN (200 µM). PBS was used as a control. After another 2 days, tumor spheroids were fixed with 4% paraformaldehyde for 15 min and permeabilized with PBS containing 0.025% Triton X-100. Samples were blocked using normal goat serum containing 5% BSA for 2 h at room temperature, then incubated with primary antibodies, including E-cadherin (1:100, Abcam), N-cadherin (1:100, Abcam), vimentin (1:100, Abcam) and FAKY397 (1:100, Abcam)
overnight at 4℃. Next day the samples were incubated with DyLight 549-conjugated secondary antibody (1:200, Abbkine, Wuhan, China) and nuclei were counterstained with DAPI (1:3000, Thermo Fisher Scientific). TRITC Phalloidin (1:200, YEASEN, Shanghai, China) staining was performed to observed cellular morphologies. Images were recorded using a Nikon confocal microscope (Nikon A1R). UM-SCC6 spheroid area and expression areas of E-cadherin, N-cadherin, Vimentin and FAKY397 were quantified with Image Pro Plus 6.0 using 5 random high-power fields of each well.
To detect collagen crosslinks, collagen I-Matrigel mixture (75 µL/well) in a 96-well plate was treated by CAF sEVs (100 µg/well) with and without BAPN (200 µM) for 12 h. Then, the levels of PYD, DHLNL, and HLNL in the mixture were quantified by ELISA assay.
UM-SCC6 cells (2×106 cells /mouse) were injected into the subcutaneous space of nude mice aged 6 weeks old (about 18 g, female). After 2 weeks, the average diameter of subcutaneous xenografts was around 5 mm. CAF sEVs (100 µg/mouse) were injected into the peritumor region every 3 days. BAPN (100 mg/kg) was injected intraperitoneally every other day. PBS was used as a control. Mice were weighed every 3 days. Xenografts were measured using calipers every week and the volumes were calculated by the formula: (width)2 × length/2. When most xenografts reached 1000 mm3 at week 6, mice were sacrificed. Xenografts were harvested and fixed in 10% formalin buffer for 24 h, embedded in paraffin. Hematoxylin and eosin (HE) staining was performed to confirm the histological features of these xenografts. Immunofluorescent staining was performed to assess EMT. Primary antibodies included LOX (1:200, Abcam), E-cadherin (1:100, Abcam), N-cadherin (1:100, Abcam), vimentin (1:100, Abcam). All animal experiments were strictly performed in accordance with animal care guidelines approved by the Animal care and Use Committee of Dalian Medical University.
GraphPad Prism 7.0 (Graphpad Software Inc.) and Image Pro Plus 6.0 was used for statistical analyses. Unpaired Student’s t-tests was performed for comparisons between all of the data. Error bars shown in graphical data represent the mean ± SD. Statistical significance was defined as p< 0.05. Each experiment was repeated at least three times.