Human tissue and blood samples
All clinical samples were collected with written informed consent from patients in the First/Third/Forth Affiliated Hospital of Harbin Medical University, and the ethical approval was granted by the Committees for Ethical Review of Harbin Medical University. Blood and original tumor specimens from 133 CCRCC patients at stages I and II (located CCRCC) and from 76 patients at stages III and IV (metastatic CCRCC) who underwent surgery. The collected tissues were immediately divided into two parts used for primary cultures and snap-frozen in liquid nitrogen, respectively. This study was approved by the Institutional Review Board of Harbin Medical University, and all subjects provided their informed consent.
Patient-derived CCRCC cells and cancer stem cells (CSCs)
CSCs were identified and isolated from human renal carcinomas as previously described . Briefly, each CCRCC tissue specimen was minced into 1 mm3 cube chunks and enzymatically dissociated to single cells. Cells were isolated, using anti-CD105 antibody coupled to magnetic beads. CD105+ cells were maintained in CCRCC stem cell culture medium. CD105− renal tumor cells were plated and maintained in DMEM with 10% exosome-depleted FBS. Finally, the cells were placed in an incubator at 37 °C with 5% CO2 and saturated humidity.
The human renal clear cell carcinoma (CCRCC) ACHN and 786-O cells, and human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (ATCC). 786-O cells were maintained in RPMI-1640 Medium. ACHN and HEK293 were maintained in DMEM medium. The medium was supplemented with 10% fetal bovine serum (Invitrogen, USA).
Exosomes isolation and application
Exosomes were isolated as described by Liu et al . Briefly, patient-derived cells were starved for 12 h (without FBS), after which the medium was collected, centrifuged at 300 g for 10 min, and 20,000 g for 20 min at 4 °C to remove cellular debris. Next, the supernatant was filtered using a 0.2 mm filter and centrifuged at 100,000×g for 90 min at 4 °C. The final pellet containing exosomes was re-suspended in PBS and the amount and size of exosomes were determined by NanoSight.
Protein content of exosomes was quantified using BCA Protein Assay. For in vitro application of exosomes, 5 × 105 cells were incubated with 50 μg exosomes, and for in vivo application of exosomes, 5 mg exosomes were intravenously injected into BALB/c nude mice (SLAC Laboratory Animal Company, China) via tail vein.
Transmission electron microscopy
Selected samples were fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate-buffered saline (PBS; pH 7.4) and fixed at 4 °C overnight. The specimens were then rinsed in buffer, post-fixed in PBS 1% OsO4 for 1–2 h. The samples were embedded in 10% gelatin and fixed in glutaraldehyde at 4 °C. They were then cut into blocks, stained en bloc in uranyl acetate, dehydrated in ethanol, and embedded in epoxy resin by standard procedures. The ultra-thin sections were electron-stained and observed under an electron microscope (JEM-1220, JEOL Ltd., Japan).
Wound healing assay
CCRCC cells were seeded in six-well plates in culture medium, and grown to 70% confluence. They were then rinsed with phosphate-buffered saline (PBS). A sterile 200 μL pipette tip was used to create wounds, and the cells were incubated with exosomes for 48 h before the assessment of cell migration across the wound line.
CCRCC cells (1 × 105) were seeded into upper chambers. The chambers were then inserted into transwell apparatus (Costar, USA). The upper chambers were coated with Matrigel (BD Biosciences, USA) when cell invasion assay was done. Medium with 10% FBS was added to the lower chamber. After 48 h, cells on the bottom of the inserts were fixed in 4% paraformaldehyde and stained with 0.05% crystal violet. Then cells that invaded into the lower surface were counted. Each experiment was repeated at least three times.
RNA extraction and quantitative real-time polymerase chain reaction
Total RNA was extracted and purified using a miRNeasy Mini Kit (Qiagen, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in triplicate in the ABI 7500 fast real-time PCR System (Applied Biosystems, USA). The relative expression level of miR-19b-3p was calculated through normalization to U6 internal controls, and mRNAs were normalized with Actin. The following primers were used for PCR detection:
5′-AGAACGCATTGCCACATACACTC -3′ (F),
5′-CATTCTGATCGGTTACCGTGATC -3′ (R)
5′-ACAGTGGCCACCTACAAAGG -3′ (F),
5′-CCGAGATGGGGTTGATAATG -3′ (R)
5′-GAGAACTTTGCCGTTGAAGC -3′ (F),
5′-GCTTCCTGTAGGTGGCAATC -3′ (R)
5′- GGAGTCCGCAGTCTTACGAG -3′ (F),
5′-TCTGGAGGACCTGGTAGAGG -3′ (R)
Protein extraction and western blot
Exosomes or cells were lysed with RIPA buffer containing a complete protease inhibitor tablet (Roche, Switzerland). Lysate was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gel was blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked in 5% nonfatmilk, and then incubated with either rabbit anti-human E-cadherin (3195, Cell Signaling Technologies, USA), N-cadherin (14,215, Cell Signaling Technologies, USA), Vimentin (5741, Cell Signaling Technologies), Twist (ab49254, Abcam, USA), PTEN (ab32199, Abcam, USA), CD103 (GTX64393, Genetex, USA), CD9 (20597–1-AP; Proteintech, USA), or Actin (3700, Cell Signaling Technology, USA). After washing, the membrane was incubated with the fluorescence-conjugated anti-mouse or anti-rabbit IgG (Invitrogen, USA). The bound secondary antibody was quantified using the Odyssey v1.2 software (LI-COR, USA) by measuring the band intensity (area × optical density) for each group and then normalized with Actin. The final results are expressed as fold changes by normalizing the data to control values.
CCRCC cells (1 × 106) suspension was subcutaneously injected into the flank of 5-week-old female athymic BALB/c nude mice (SLAC Laboratory Animal Company, China). Meanwhile, a dosage of 5 mg exosomes administered into mice via tail vein injection once every 3 days for 2 weeks. Growth rate of tumors was determined by measuring tumor size at fixed time points as to be specified. Tumor size was measured with calipers after the tumor cell injection once every 7 days for a total period of 4 weeks. Tumor volume was determined using the formula: volume = length × width2/2.
To evaluate metastasis, cells (stable luciferase transfected CCRCC cells) were injected into nude mice through tail vein. Tumors derived from stable luciferase-transfected cells were imaged to observe luciferase expression on day 28 after tumor cell injection. Briefly, the animals were anesthetized and then injected (i.p.) with luciferin at 150 mg/kg in a volume of 100 μL. Images were captured at a peak time of 15–20 min after injection using an IVIS-200 Imaging System (Xenogen Corporation, USA).
For in vivo exosome-tracking experiments, purified exosomes were fluorescently labeled with PKH26 membrane dye (Sigma, USA). Labeled exosomes (1 mg) were intravenously injected into mice. Organs were collected at 12 h after exosome injection and analyzed using Living Image software (Xenogen Corporation, USA).
All animal experiments were undertaken in accordance with the NIH Guide for the Care and Use of Laboratory Animals, with the approval of the Institutional Animal Care and Use Committee of Harbin Medical University.
Statistical analysis was performed with SPSS13.0 software. Student t test, ANOVA, or chi-square analysis was applied, where appropriate. Survival rates were estimated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. A probability of < 0.05 (*) or < 0.01 (**) was considered significant.