Specimens and primary cells
Human serum specimens and liver tissues were collected from healthy donors and HCC patients before or after resection in the Nanjing Drum Tower Hospital in Nanjing, China. All procedures were approved by the Ethical Committee of the Nanjing Drum Tower Hospital. Written informed content was obtained from every participant prior to study.
Cell culture
The human liver cancer cell lines (97H, LM3 and Huh7), the vascular endothelial cell line HUVEC, and the HSC cell line LX2 were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology) and maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco). The normal liver cell line LO2 was purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco). All cell lines were cultured at 37 °C in a humidified incubator containing 5% CO2. Cell lines were authenticated by short tandem repeats (STR) profiling and confirmed to be mycoplasma negative.
Reagents and antibodies
Antibodies against CD63 (ab125011, 1:1000), α-SMA (ab32575, 1:100), FAP (ab207178, 1:1000), FSP (ab124805, 1:1000), GAPDH (ab8245, 1:10,000), CD9 (ab92726, 1:1000), CD81 (ab79559, 1:1000), FASN (ab22759, 1:500), ATP citrate lyase (EP704Y, 1:1000) and phospho-ATP citrate lyase (T447/S451) (ab53007, 1:1000) and USP2 (ab66556, 1:500) were purchased from Abcam (Cambridge, MA, USA). Antibodies against phosphor-PTEN (Ser380/Thr382/383) (9554S, 1:1000), total PTEN (7960 T, 1:1000), PDK1 (3062 T, 1:1000), phospho-PDK1 (Ser241) (3438 T, 1:1000), phospho-Akt (Ser473) (4060 T, 1:1000) and AKT (4691 T, 1:1000) were supplied by Cell Signaling Technology (Beverly, MA, USA). AKT inhibitor MK-2206 was provided by Selleck (Houston,USA).
Western blotting analysis
Whole-cell protein extracts were homogenized in lysis buffer and centrifuged at 12,000 r.p.m. for 15 min. Protein concentrations were determined by bicinchoninic acid (BCA) assay. After immunoblotting, the proteins were transferred onto nitrocellulose membranes, followed by incubation with specific antibodies. The immunocomplexes were then incubated with the fluorescein-conjugated secondary antibody, and immunoreactive bands were visualized by an Odyssey fluorescence scanner (Li-Cor, Lincoln, NE).
RNA interference
siRNAs and mimics of indicated miRNAs were obtained by RiboBio Company (Guanngzhou, China). The sequences of siRNAs and miRNA mimics referred above were listed in Additional file 1: Table S1. Transfection with siRNAs and miRNAs was completed using riboFECT™ CP (RiboBio) according to the manufacturer’s instructions.
Animal studies
To examine the roles of exosomes in CAFs, 1 × 106 Huh7 cells were intravenously injected into male nude mice through the tail vein (Chinese Science Academy, Shanghai, China). Subsequently, mice were randomly divided into groups and intravenously injected with equal numbers of exosomes from different tumor cells twice a week for 1 month. For xenograft assays, 1 × 106 Huh7 cells and 1 × 106 LX2 cells were injected subcutaneously into the right side of each male nude mouse (Chinese Science Academy). The sizes of tumors (length × width: 2 × 0.5) were measured at the indicated time points, and tumors were obtained at 4 weeks after injection. All animal experiments were approved by the University Committee on Use and Care of Animals of Nanjing Drum Tower Hospital.
Cell viability assay, migration assay and wound-healing assay
For cell viability assay, Cell Counting Kit 8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) was used to assess cell viability according to manufacturer’s instructions. For migration assay, 5 × 104 LX2 cells were plated into 24-well transwell plates with inserts (Corning). The medium in inserts was free of FBS, whereas the medium outside the inserts was supplemented with 10% FBS. To detect exosome function, equal quantities of tumor-derived exosomes were added into the inserts. After 24 h, the cell inserts were fixed and stained according to manufacturer’s protocols. Representative fields were photographed, and the number of migrated cells per field was counted. For wound-healing assay, equal numbers of LX2 cells were plated into six-well plates. Then the cell monolayers were wounded with a pipette tip to draw a gap on the plates. After treated with tumor-derived exosomes, fibroblasts that migrated into the cleared section were observed under microscope at the specific time points.
RNA extraction and qRT-PCR
HSC RNA was extracted from snap-frozen liver tissues with TRIzol™ reagent (Life Technologies, USA) according to the manufacturer’s instructions. Briefly, cells in six-well plate were homogenized in 1 mL TRIzol™ reagent at room temperature. Then 200 μL trichloromethane was added and mixed thoroughly, followed by centrifugation at 12,000×g for 15 min at 4 °C. The upper clear phase was collected and added with 500 μL isopropanol, followed by centrifugation at 12,000×g for 10 min at 4 °C. The liquid supernatant was discarded, and the pellet was washed with 75% ethanol. The RNA was re-suspended in 50 μL RNase-free DEPC-water and stored at − 80 °C.
Reverse transcription was performed with PrimeScript™ RT Master Mix (Takara, Japan) according to the manufacturer’s instructions. qRT-PCR was performed using TB Green™ Premix Ex Taq™ (Takara, Japan) on an ABI PRISM 7500 real-time PCR System (Applied Biosystems, USA). Primers used for qPCR are shown in Additional file 1: Table S1. The relative expression levels of mRNAs were calculated with 2–ΔΔCt method. GAPDH was selected as the housekeeping gene.
miRNeasy Mini Kit (Qiagen) was used to extract RNA from exosomes in plasma/medium according to the manufacturer’s instructions, and cel-miR-39 (Takara) was added into each sample at a final concentration of 10 pmol/μL acting as external reference. Total RNA was stored at − 80 °C for subsequent experiments.
Reverse transcription and qRT-PCR for exosomal miRNA, as well as internal reference U6 were performed using miRNA RT-PCR Quantitation Kit (Qiagen) according the manufacturer’s instructions. Briefly, after an initial denaturation step at 95 °C for 3 min, the amplifications were carried out with 40 cycles at a melting temperature of 95 °C for 15 s, and an annealing temperature of 62 °C for 34 s. The relative expression levels of exosomal miRNAs were calculated with 2–ΔΔCt method.
Oil red staining
Cells were fixed in 10% formalin, washed with 60% propylene glycerol, and then stained with 0.5% oil red O (Sangon Bio) in propylene glycerol for 10 min at 60 °C. The red lipid droplets were visualized by microscopy.
Immunohistochemistry and in situ hybridization analysis
For immunohistochemistry, the slides were incubated with above-mentioned primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Finally, the staining processes were performed with diaminobenzidine colorimetric reagent solution (Dako, Carpinteria, USA) and hematoxylin (Sigma Chemical Co., USA). For in situ hybridization analysis, hsa-miRNA-21 miRCURY LNA detection probe (Exiqon, Denmark) was used, and the total staining processes were carried out according to manufacturer’s protocols. Images were captured with Aperio ScanScope AT Turbo (Aperio, USA) and assessed with image-scop software (Media Cybernetics, Inc.).
Immunofluorescence analysis
Immunofluorescence analysis was performed according to previously established protocols. Cells were seeded into 24-well dishes and fixed by 4% paraformaldehyde 24 h later. Fixed cells were stained with α-SMA and FAP antibodies (mentioned above), followed by incubation with FITC-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG (Abcam). Representative images were detected by fluorescence microscopy (Leica, German), and data were processed via Photoshop.
Tube formation assay
EC matrix was thawed at 4 °C overnight, and required wells of pre-chilled 96-well plates were coated with 50 μL diluted EC matrix and incubated at 37 °C for 1 h to solidify. Subsequently, 150 μL of HUVEC cells (1 × 104) with different conditional media were added to the solidified matrix and incubated at 37 °C for 12 h. Endothelial cell formation was observed using a microscope. Focus was placed on distinct areas, and the tubes formed were counted according to the kit procedure.
Isolation and analysis of exosomes
For exosome isolation, equal numbers of different cells were transplanted into 10-cm plates and maintained in fresh DMEM supplemented with serum, which was depleted of exosomes by centrifugation at 12,000×g overnight. After 48 h, CM was collected and filtrated through 0.22-μm filters (Millipore, USA). Exosomes in CM or serum samples were isolated by ultracentrifugation according to the standard methods described previously [22]. Ultracentrifugation experiments were performed with Optima MAX-XP (Beckman Coulter, USA). Exosomes were observed by Philips CM120 BioTwin transmission electron microscope (FEI Company, USA).
Exosomes tracing
For exosome-tracing experiments, tumor cells were pre-treated by PKD67 (Beyotime, China), and exosomes in CM were obtained as described above. After incubation with recipient cells that were pre-treated with FAP antibody and DAPI (Beyotime), exosomes were observed by fluorescence microscope (Leica, German).
Transmission electron microscopy
The obtained exosomes were fixed with fixative buffer containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PBS. After embedding, samples were cut into 0.12-μm sections and stained with 0.2% lead citrate and 1% uranyl acetate. The images were detected by a JEOL TEM-2000 EX II (JEOL, Tokyo, Japan).
Cell cycle analysis
HSCs were seeded into six-well plates at a density of 1.5 × 105 cells/well overnight, and the cells were conditionally cultured for 24 h. Cell cycle was detected using cell cycle analysis kit (Thermo Fisher Scientific, USA) according to the manufacture’s protocols and measured by flow cytometry.
Collagen contraction assay
A total of 2 × 105 HSCs were suspended in 100 μL DMEM. Then the cell suspension was mixed with 100 μL of collagen mix containing 68.75 μL DMEM, 0.72 μL 1 N NaOH and 31.25 μL type 1 rat tail collagen (Corning), added to one well of 6-well plates and allowed to solidify for 45 min at 37 °C. After incubation with medium containing HCC-derived exosomes, the gels were photographed by digital camera.
Statistical analysis
Data analysis was performed using the SPSS software version 16. Each experiment was carried out in triplicate at least, and all results were presented as mean ± SD. χ2-Test and Student’s t-test were used to assess statistical significance. Kaplan–Meier analysis and log-rank tests were applied for survival analysis. A p value of < 0.05 was considered as statistically significant.