Purchased cell lines
The HEp-2 cell line (human laryngeal carcinoma cells) was purchased from the American Type Culture Collection (ATCC). The HEK-293 cell line (human embryonic kidney 293 cells) and SK-OV-3 cell line (human ovarian epithelial cancer cells) were purchased from the Cell Bank of the representative culture preservation committee of the Chinese Academy of Sciences (Shanghai, China). The HCT116 cell line (human colorectal carcinoma cells) and the MDA-MB-231 cell line (human breast cancer cells) were kindly provided by Professor Jun Du (Sun Yat-sen University, Guangzhou, China). HEp-2 cells were cultured in DMEM (high-glucose) supplemented with 5% (vol/vol) fetal bovine serum (FBS). HEK-293 and MDA-MB-231 cells were maintained in DMEM (high-glucose) containing 10% (vol/vol) FBS. SK-OV-3 cells were cultured in McCoy’s 5A medium supplemented with 10% (vol/vol) FBS. All culture media contained 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37°C.
Generation of stable cell lines
The stable cell line 293-miR-HER2, expressing miRNA targeting HER2, was generated by transfection of the miR-HER2-E1 plasmid into HEK-293 cells. Forty-eight hours after transfection, cells were selected by the addition of blasticidin (Solarbio Life Sciences) to a final concentration of 6 μg/ml. A cell colony with green fluorescent protein (GFP) expression was selected and cultured in complete medium with 6 μg/ml blasticidin. The cell line was monitored for the expression of GFP and miR-HER2-E1.
To generate a cell line producing exosomes that adhere to the surface of HER2-positive cells, 293-miR-HER2 cells were either infected with lentivector XSTP724PA-1 (XStamp HER2 ligand exosome HER2 receptor targeting lentivector) or infected with control lentivector XSTP710PA-1 according to the manufacturer's instructions (XStamp Technology, SBI: XSTP724PA-1/XSTP710PA-1). The two cell lines were renamed 293-miR-XS-HER2 and 293-miR-XS (control), respectively. The lentivector XSTP724PA-1 contains two copies of the HER2 ligand fused to the 5’ N-terminal signal sequence leader and fused in frame to the 3’ C-terminal C1C2 XStamp domain that directs the entire fusion protein to be displayed on the surface of secreted exosomes [27, 28]. The HER2 binding ability of the exosomes purified from 293-miR-XS-HER2 cells was confirmed by ELISA.
Antibodies
The anti-HER2 (Cat. No. #2165S) antibody was purchased from Cell Signaling Technology. The anti-GAPDH, anti-Alix, anti-CD9, anti-Annexin V, anti-Flotillin-1, and anti-TSG101 antibodies have been described elsewhere [29].
Plasmid construction
The miRNA sequences targeting the HER2 gene were designed using BLOCK-iT™ RNAi Designer (Life Technologies) and synthesized by Ige Biotechnology (Guangzhou, China). The synthesized miRNA fragments were digested with the BamHI and XhoI restriction enzymes and cloned into the corresponding sites in the pcDNA6.2-GW/EmGFP-miR-neg control plasmid (Invitrogen). The miRNA sequences were as follows:
miR-HER2-1: 5'-AACTCAAGCAGGAAGGAAGGTGTTTTGGCCACTGACTGACACCTTCCTCTGCTTGAGTT-3'
miR-HER2-4: 5'-TGTGAGAGCCAGCTGGTTGTTGTTTTGGCCACTGACTGACAACAACCATGGCTCTCACA-3'
miR-HER2-E1: 5'-AACTCAAGCAGGAAGGAGGAGGTTTTGGCCACTGACTGAC CTCCTCCTCTGCTTGAGTT-3'
miR-HER2-E4: 5'-TGTGAGAGCCAGCTGGAGGAGGTTTTGGCCACTGACTGACCTCCTCCATGGCTCTCACA-3'
The sequences of the mature miRNAs are underlined.
miR-HER2-E1 and miR-HER2-E4 were modified versions of miR-HER2-1 and miR-HER2-4, respectively, as they contained EXO-motifs).
The HER2 expression plasmid containing the 3’-UTR sequence of the HER2 gene was generated as follows. First, the His-tagged HER2 coding sequence was amplified by PCR using the following primers:
Forward, 5’-CCCAAGCTTATGGAGCTGGCGGCCTTGTG and
Reverse, 5’-ATAAGAATGCGGCCGCTTATCAGTGATGGTGATGGTGATGCACTGGCACGTCCAGACCCAG.
The PCR fragment was then subcloned into pcDNA3.1(+) at the HindIII/NotI sites to generate pHER2-His. The 3’-UTR sequence was synthesized by Ige Biotechnology (Guangzhou, China), digested with the NotI and XbaI restriction enzymes and cloned into the corresponding sites in pHER2-His.
Exosome isolation and quantification
HEK-293 cells (1 × 107) seeded in a T150 flask were mock transfected or transfected with 10 μg of the NT or miR-HER2-E1 plasmid. After 4 h of incubation, the cells were rinsed extensively with phosphate-buffered saline (PBS) and incubated in serum-free medium for an additional 48 h. Cells of the stable cell lines were seeded in a T150 flask for 24 h, rinsed extensively with PBS and incubated in serum-free medium for another 48 h. Cell-free extracellular medium containing exosomes was harvested by centrifugation at 300 × g for 10 min to remove the cells. The supernatant was then centrifuged at 10,000 × g for 30 min to remove dead cells and cell debris. Finally, the clear supernatant was centrifuged for 70 min at 100,000 × g to pellet the exosomes. All centrifugation steps were carried out at 4°C. For immunoblotting, the pelleted exosomes were resuspended in RIPA buffer. For treatment of cells or mice, the pelleted exosomes were resuspended in PBS. The exosomes were quantified by the BCA method using an Enhanced BCA Protein Assay Kit (Beyotime) according to the manufacturer's instructions.
Exosome size analysis
The size distribution of the exosomes was analyzed using the Izon qNano system (Izon, Christchurch, New Zealand; www.izon.com), which uses single-molecule electrophoresis to detect extracellular vesicles passing through a nanopore. The procedure yielded accurate particle-by-particle enumeration of exosomes ranging from 75 to 300 nm in diameter. Specifically, purified exosomes were diluted 1:10 in PBS containing 0.05% Tween 20, shaken vigorously, and measured by using an NP200 (A53942) nanopore aperture according to the manufacturer’s instructions. The results were analyzed using Izon Control Suite software v3.3 (Izon Science).
Quantitative RT-PCR for miRNA
Total RNA from cells and suspended exosomes was isolated using TRIzol reagent (Thermo Fisher Scientific) and TRIzol LS reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The miRNAs tested were reverse transcribed from 50 ng of total RNA in duplicate with specific stem-loop primers as described in the TaqMan miRNA reverse transcription kit instructions (Applied Biosystems, Inc.). miRNA expression was measured by real-time PCR using a TaqMan Universal Master Mix II kit purchased from Applied Biosystems, Inc. The miRNA copy number was normalized to that of cellular 18S rRNA. The primers specific for miR-HER2-E1 were designed according to Chen et al [30] and synthesized by Ige Biotechnology. The sequences follow:
miR-HER2-E1 stem-loop primer,
5’-GTCGGTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCCTCCT-3’;
Forward primer, 5’-AACCAAGCAGGAAGGAGG-3’;
Reverse primer, 5’-GTGCAGGGTCCGAGGT-3’;
Probe, 5’-(6-FAM) TCGCACTGGATACG (MGB)-3’.
Plasmid transfection
SK-OV-3 or HEp-2 cells were seeded in 12-well plates at 2.5 × 105 cells per well. Cells were transfected with 0.5 μg of plasmids expressing miR-HER2-E1, miR-HER2-E4 or non-targeting (NT) miRNA or for HEp-2 cells were contransfected with 0.2 μg of a plasmid encoding His-tagged HER2 using Lipofectamine 2000 reagent (Thermo Fisher Scientific) following manufacturer's instructions. Cells were harvested at 72 h after transfection and used for immunoblotting analysis.
Exosomes incubation
SK-OV-3 cells at 2.5 × 105 cells per well were exposed to different concentrations of purified exosomes (0.1 mg, 5 mg, or 20 mg) carrying miR-HER2-E1 or purified exosomes (20 mg) produced by NT-transfected HEK-293 cells or remain no treatment (Con) for 72 h. For HEp-2 cells at a density of 2.5 × 105 per well were transfected with 0.2 μg of a plasmid encoding His-tagged HER2 for 36 h and then incubated 20 µg of purified exosomes produced by HEK-293 cells transfected with plasmids encoding the miR-HER2-E1 or the NT miRNAs or remain no treatment for another 36 h. The cells were then harvested for endogenous and exogenous HER2 expression by immunoblotting analysis.
Immunoblotting assay
Cell pellets or purified exosomes were harvested and lysed with RIPA lysis buffer (Beyotime) supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (1 mM; Beyotime) and a phosphatase inhibitor (Beyotime). Cell lysates and exosomes were heat denatured at 100℃ incubator for 10 min, separated by 10% or 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The proteins were identified by incubation with the appropriate primary antibody and then with an HRP-conjugated secondary antibody (Pierce). Immunoreactions were visualized with ECL reagent (Pierce), and images were acquired using a ChemiDoc Touch Imaging System (Bio-Rad) and analyzed with Image Lab software. The densities of the corresponding bands were quantified using ImageJ software.
Cell viability assay
SK-OV-3, HCT116, HEp-2 and MDA-MB-231 cells were seeded into 96-well plates at a density of 1 × 104 cells per well one day before exposure to exosomes. Cells in triplicate wells were mock treated (Mock) or incubated with 1 µg of purified exosomes produced by HEK-293 cells transfected with plasmids encoding either miR-HER2-E1 or the nontargeting (NT) miRNA. After 72 h of incubation, the relative cell viability was determined by a CCK8 assay according to the manufacturer’s protocol.
Animal models
BALB/c nude mice at 6-7 weeks of age were purchased from Vital River Laboratory Animal Technologies Co., Ltd. (Beijing, China). The nude mice were injected subcutaneously in the flanks with 5 × 106 SK-OV-3, HCT116 or MDA-MB-231 cells respectively. In the exosome-delivered miR-HER2-E1 treatment, mice with tumors having an average volume of 90 mm3 were injected intratumorally with 50 ml containing 10 μg of purified exosomes per injection. Each tumor-bearing animal was injected on days 1, 4, 7, 10, 13, and 16, for a total of 6 injections. The size of tumors was measured on days 1, 4, 7, 10, 13, 16, 19 and 22. In the exosome-delivered 293-miR-XS-HER2 treatment nude mice derived from BALB/c were injected subcutaneously into flanks with 5 × 106 SK-OV-3 cells. Tumors averaging 80 mm3 were injected intravenously on days 1, 4, 7, 10, 13, 16, 19 and 22 with 3 μg/animal or 30 μg/animal of exosomes purified from 293-miR-XS-HER2, 293-miR-XS or parental HEK-293 cells. The sizes of tumors were measured on days 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28 using a caliper, and the volume was calculated as (length × width2) × 0.5.
ELISA
Exosomes purified from the 293-miR-XS or 293-miR-XS-HER2 stable cell lines were coated (1 μg/well) in triplicate wells of 96-well ELISA plates (Corning). After the coating solution was removed, nonspecific binding sites were blocked by incubation with 2% BSA at 37°C for 1 h. The plates were rinsed, exposed to HER2 protein (Sino Biological, China) for 2 h, rinsed again, and reacted with the HRP-conjugated rabbit anti-HER2 antibody (Cat. No. 1004-R205, Sino Biological) for an additional 1 h. The plates were then rinsed again and exposed to TMB for color development. The reaction was terminated by the addition of stop solution. The plates were read in a BioTek microplate reader at a wavelength of 450 nm.