SACC-83 and SACC-LM were kind gifts from Peking University. Primary CAF cells, CAF-A1 and CAF-A2, were isolated from fresh tumor tissues of two SACC patients as described previously. All of cells were cultured in Dulbecco modified Eagles medium (DMEM)/F12 (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; ScienCell, USA), 100U/ml penicillin, and 100U/mL streptomycin (Gibco) with 5% CO2 at 37 Cº.
CAF cells were cultured in 75cm2 flasks till cell confluency reached more than 80%. Then, they were washed with PBS and incubated with serum-free DMEM/F12 for 48 hours. The supernatant was collected as condition medium (CM) and stored at -80 Cº until use. CM was sequentially centrifuged at 500 g, 2,500 g and 10,000 g and the supernatant was collected. Then EVs were isolated from the supernatant using Total Exosome Isolation Reagent (Invitrogen 4478359).
Transmission electron microscopy (TEM)
EVs were evaluated for their morphology and size by TEM. Briefly, 40 µL of EV samples were placed on a film, then copper mesh is applied on it for 15 min, after that the copper was drained with filter paper and 3% of phosphotungstic acid was put on the mesh for 5 minutes. Finally filter paper was used to drain the negative dye solution and filter the mesh. Then the sample was observed under the electron microscope JEM-2000EX9 (JEOR, JAPAN).
Wound healing assay
The migration abilities of both SACC-83 and SACC-LM cells were assessed by wound healing assay. Cells were seeded in 6-well plate till 100% confluency, then a straight scratch was made by using P1000 pipette tip. Then cells were washed with PBS and cultured with CAF EVs (10 µg/well) in serum free DMEM/F12 for another 48 hours. DMEM/F12 without EVs was used as a negative control. Images were recorded by an inverted fluorescence microscope (Olympus IX71). The gap closure was evaluated as the cell migration rate. The relative closure rate of each sample was measured using Image-Pro Plus 6.0.
Transwell invasion assay
We assessed the invasion ability of both SACC-83 and SACC-LM cells by transwell invasion assay. It was performed using the transwell chamber consisting of 6.5 mm diameter inserts (Corning Inc., USA). The membrane with 8.0 µm-pore-size was coated with 1:10 diluted Matrigel (Corelle Life Science Co., Ltd). Briefly, SACC-83 and SACC-LM cells were seeded in the upper chamber and CAF EVs (10 µg/well) was added to the lower chamber for the induction. After 48 hours, the non-invading cells in the upper chamber were removed by cotton swaps and the cells that invaded to the bottom chamber were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet (coolaber science and technology), and photographed with an inverted microscope (Olympus IX 71). Finally, we took images from five random fields and counted the cells in each chamber to get the invaded cell number as the mean. The data represent at least three experiments (mean ± standard error).
Protein was extracted using RIPA buffer (Anzure Sky Biological Technology Co., Ltd.) and protein concentration was detected by BCA assay kit (Beyotime Biotechnology). Subsequently, protein (20 µg) from each sample was separated by 12% SDS-polyacrylamide gel, transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (PIERCE), and probed with primary antibodies, including E-cadherin 1:4000 (20874-1-AP, Proteintech.), N-cadherin 1:800 (18203, Abcam), vimentin 1:1000 (10366-1-AP, Proteintech.), IL-6 1:2000 (9324, Abcam), CD81 1:200 (BM14579), CD63 1:200 (25682-1-AP, Proteintech.), HSP70 1:500 (32239, Santa Cruz), Calnexin 1:500 (10427-2-AP, Proteintech.), JAK2 1:800 (17670-1-AP, Proteintech.), STAT3 1:4000 (10253-2-AP, Proteintech.), p-JAK2 1:1000 (“phospho Y1007 + Y1008” 32101, Abcam), p-STAT3 1:2000 (“phospho S727” 32143, Abcam), and GAPDH 1:4000 (10494-1-1AP, Proteintech.). The membranes were blocked with skimmed milk (Becton, Dickinson, USA). Proteins were detected by horseradish peroxidase-conjugated antibody and goat anti-rabbit IgG (Boosten Company) at 1:2000 and 1:4000 dilution respectively with 1% BSA-PBS (including 0.05% TWEEN20) and revealed using the ECL system (Advantsa).
RNA interference transfection
IL6-specific siRNA (si-IL6: TTCGGTCCAGTTGCCTTCT) was used to knockdown IL6 expression. A non-targeting siRNA (siNC: CGUACGCGGAAUACUUCGA) was used as a negative control. These products were purchased (RiboBio, China). These siRNA at the final concentration of 5 nmol/250 µL were transfected into CAF cells with Lipofectamine 2000 reagent (Invitrogen, USA).
Subcutaneous xenograft model
The use of BALB/c nude mice (3-4 weeks, female) were approved by the Institute Animal Care and Use Committee of Dalian Medical University. Animal experiments, transportation, and care were conducted in compliance with the relevant laws and the guidelines issued by the Ethical Committee of Dalian Medical University. Mice were divided into three groups with five mice in each group: SACC-LM group was injected with 2.25 × 106 cells/mouse; SACC-LM+CAF-A1 group was injected with 2.0 × 106 SACC-LM and 2.5 × 105 CAF-A1 per mouse; SACC-LM+CAF-A2 group was injected with 2.0 × 106 SACC-LM and 2.5 × 105 CAF-A2 per mouse. Cells were resuspended in 50µL PBS and 50µL Matrigel, then injected subcutaneously. The mice were raised for 5 weeks. Mice were anesthetized with 20% urethane when maximum tumor diameter reached to 15 mm. After euthanizing, the tumors were harvested and fixed with 4% paraformaldehyde and 30% sucrose solution overnight, then embedded into OCT and prepared into sections.
Immunohistochemical staining of xenografts was performed on 8-µm-thickness sections using SPlink Detection Kits (SP-9000, ZSGB-BIO, China). The sections were rinsed with PBS and incubated with 3% hydrogen peroxide in methanol for blocking endogenous peroxidase activity. The nonspecific binding sites were blocked with 10% goat serum for 30 minutes. Sections were incubated with anti-human pan-CK (Merck Millipore), Vimentin (1:200, Proteintech), IL-6 (1:200, Proteintech), E-cadherin (1:200, Proteintech), N-cadherin (1:200, Abcam) overnight at 4°C. Immunoreactions were detected using 3, 3ʹ-diaminobenzidine. Nuclei were counterstained with hematoxylin. The integrated optical density and area of target distribution were measured with Image-Pro® Plus version 6.0.
Statistical analysis was performed using SPSS 13.0 software. The one-way analysis of variance (ANOVA) is used to determine any statistically significant differences. Significance was defined at p< 0.05.