Cell lines and culture conditions
Three human gastric cancer cell lines (MKN-28, MKN-45, and MKN-74) were purchased from Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan) and SW480, a human colorectal cancer cell line, was obtained from American Type Culture Collection (ATCC, USA). MKN-28 and its transfectants, MKN-45 and MKN-74, were maintained in Dulbecco’s modified Eagle medium (DMEM; 05919, Nissui Pharmaceuticals, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; 1370978, Gibco, Gaithersburg, MD, USA), P.S. (100 Units/mL penicillin and 100 µg/mL streptomycin, Gibco), and L-glutamine (30 mg/mL, Nacalai Tesque, Kyoto, Japan). SW480 cells were cultured in Roswell Park Memorial Institute 1640 medium (13485; Gibco) supplemented with 10% FBS. Human hepatic sinusoidal endothelial cells (HHSECs) were purchased from ScienCell Research Laboratories (Cat. No. 5000) and were maintained in endothelial cell medium (1001; ScienCell) in bovine plasma fibronectin (10%; 8284; ScienCell)-coated dishes. All cell lines were cultured at 37°C in an atmosphere of 95% air and 5% CO2.
Plasmid transfection
Human AMIGO2 expression vector (pEZ-M02/AMIGO2) and control vector (pEZ-M02) were obtained from GeneCopeia (Rockville, MD, USA). These plasmids were transfected into MKN-28, an AMIGO2-lacking cell line, in order to establish AMIGO2-overexpressing MKN-28 cell lines (MKN-28 A1 and A2) and control lines (MKN-28 E1 and E2), respectively. MKN-28 cells were transfected with 1 µg pEZ-M02/AMIGO2 or its corresponding negative control (pEZ-M02) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol.
Isolation of sEVs
MKN-28 cells and transfectants were cultured until they reached 80% confluency in 10% FBS DMEM; the medium was then replaced with serum-free advanced DMEM (12491-015; Gibco) containing L-glutamine. After 48 h, the supernatant was collected and centrifuged at 2,000 × g for 10 min at 4°C to remove cells and cell debris. The supernatant was filtered through a 0.22 µm membrane filter (Millipore, Billerica, MA, USA). The filtered supernatant was centrifuged at 210,000 × g for 70 min at 4°C using a Beckman Coulter Optima XPN-80 ultracentrifuge with an SW41Ti rotor. After centrifugation, the supernatant was removed, and the pellet was resuspended in phosphate-buffered saline (PBS) centrifuged at 210,000 × g for 70 min at 4°C. Subsequently, PBS was removed, and the pellet containing the sEVs was resuspended in PBS. The protein concentration in the sEVs was measured using a Micro BCA Protein Assay Kit (23235; Thermo Fisher Scientific). The number of sEVs was measured using NanoSight NS300 (Malvern Panalytical, Malvern, England). Videos were recorded five times per sample for 1 min and analyzed using nanoparticle tracking analysis software (version 3.1, Malvern Panalytical).
Real-time polymerase chain reaction (PCR)
Total RNA was extracted from cells using TRIzol reagent (15596026; Thermo Fisher Scientific), and 1 μg of total RNA was reverse transcribed into cDNA using the TaKaRa PrimeScript RT master mix (TaKaRa Bio, Otsu, Japan). Five microliter of cDNA was used for performing quantitative PCR. Quantitative PCR was performed on a Roche LightCycler 480 (Roche Diagnostics, Palo Alto, CA, USA) using SYBR Premix Ex Taq II (Tli RNaseH Plus; TaKaRa Bio). The cycling program involved heating at 50°C for 2 min, 95°C for 10 min, followed by 35 cycles at 95°C for 15 s 60°C for 1 min. β-actin was used as the internal control. The following primers were used: AMIGO2-F: 5′-CCCCTGCAAGTGTAAAACCA-3′, AMIGO2-R: 5′-AGGGGTTCCAAAAACACCAC-3′; β-actin-F: 5′-AGAGGGAAATCGTGCGTGAC-3′, β-actin-R: 5′-CAATAGTGACCTGGCCGT-3′.
Western blotting
Cells were lysed in ice-cold lysis buffer (1% NP-40, 50 mM Tris (pH 7.5), 165 mM NaCl, 10 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin). Proteins were separated on 10% (CD63 and AMIGO2) or 12% (CD9) sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (ISEQ00010; Merck Millipore, Darmstadt, Germany). The membranes were blocked using 2.5% skim milk (prepared in T-PBS) at room temperature (RT) for 2 h. The membranes were then probed with the following primary antibodies: mouse monoclonal anti-AMIGO2 antibody (1:10,000; rTNK1b012a), mouse monoclonal anti-β-actin antibody (1:5,000; A5441; Sigma Aldrich, St. Louis, MO, USA), mouse monoclonal anti-CD9 antibody (1:2,500; 12A12; Cosmo Bio, Tokyo, Japan), or mouse monoclonal anti-CD63 antibody (1:5,000; 8A12; Cosmo Bio) overnight at 4°C. The membranes were then incubated with a goat polyclonal anti-rat IgG horseradish peroxidase-conjugated antibody (1:10,000; ab98425; Abcam, Cambridge, United Kingdom) or a goat polyclonal anti-mouse IgG antibody (1:5,000; PM009-7; MBL, Nagoya, Japan) at RT for 20 min. Protein signals were detected using an enhanced chemiluminescence kit (RPN2232; GE Healthcare; Buckinghamshire, United Kingdom) and analyzed using ChemiDoc Touch MP (Bio-Rad, Hercules, CA, USA).
Incorporation of sEVs into HHSECs
HHSECs (3 × 104 cells) were seeded in 24-well plates (3526, Corning, Corning NY, USA), incubated for 24 h, and then treated with 5 µg of sEVs at 37°C. sEVs were labeled using a PKH67 green fluorescent kit (MIDI67-1KT; Sigma Aldrich). sEVs were incubated with 4 µM PKH67 for 6 min at RT and then washed with PBS to remove free PKH67 via centrifugation at 14,000 × g for 2 min (five washes). After 6 h, sEVs incorporated into HHSECs were observed under a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan).
Immunofluorescence
HHSECs (3 × 104 cells) were seeded in Lab-Tek 8-well chamber slides (177402; Nunc, Rochester, NY, USA) and incubated for 48 h. Next, cells were treated with sEVs (10 µg/well) for 6 h. sEVs were labeled using a PKH red fluorescent kit (MINI26-1KT; Sigma Aldrich). HHSECs were then fixed with 4% paraformaldehyde (163-20145; Wako, Osaka, Japan) and treated with 10% normal goat serum (426042; Nichirei, Tokyo, Japan). Then, HHSECs were probed overnight with the primary antibody, i.e., anti-AMIGO2 (1:1,000, rTNK1b012a) at 4°C, followed by incubation with anti-rat IgG H&L Alexa Fluor 488 (1:5,000; ab150157; Abcam). Nuclei were stained with 4′,6-diamidino-2-phenylindole (340-07971; Dojindo, Kumamoto, Japan).
Cancer cell-endothelial cell adhesion assay
The cancer cell-HHSEC adhesion assay was performed in accordance to a previously reported method with slight modifications [5]. Briefly, a 96-well plate (165305; Thermo Fisher Scientific) was coated with fibronectin for 2 h. A total of 3 × 104 HHSECs were seeded per well, cultured for 72 h, and then treated with sEVs (1.5 µg) for 6 h. Cancer cells (2 × 105) labeled with PKH67 green fluorescent dye (PKH67GL-1KT, Sigma Aldrich) were then plated onto HHSECs and further cultured for 30 min. Non-adherent cancer cells were removed by washing with saline solution (1326; Otsuka, Tokyo, Japan), and the adherent cancer cells were quantified using a fluorescence plate reader (Infinite M200 PRO; Tecan, Männedorf, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The percentage of adherence was calculated as the fluorescence ratio, i.e., (post-wash fluorescence/pre-wash fluorescence) × 100.
Cell proliferation assay
HHSECs (3 × 103) were seeded in 96-well plates (3595; Corning) and incubated for 24 h, after which they were treated with 0.075 µg sEVs. After 24 and 48 h of incubation, HHSECs were fixed with 5% glutaraldehyde, dried, and stained using 0.5% crystal violet for 5 min. After washing with running water, cells were left to air dry. Crystal violet was eluted using 10% acetic acid and quantified by measuring the absorbance at 495 nm using a fluorescence plate reader (Infinite M200 PRO, Tecan).
Wound-healing assay
A wound-healing assay was performed to measure cell migration activity as described by Taniguchi et al. [33]. HHSECs (3 × 104) were seeded in 24-well plates (3526; Corning) and incubated for 5 days until the cells formed a monolayer. Wounds were created by scratching the cells with 200 µL pipette tips, and the medium containing the non-adherent cells was removed. At 0 h and 15 h after treatment of HHSECs with sEVs (1.5 µg), the scratched area was observed using a phase-contrast microscope (BZ-X710, Keyence). Cell migration was determined as the rate of cells moving to the scratched area, which was quantified using ImageJ (version 1.53, National Institutes of Health, Bethesda, MD, USA).
Statistical analyses
All data are presented as the mean ± standard deviation. The significance of the differences was assessed using the chi-square test and Student’s t-test. P<0.05 was considered significant.