Cell lines and cell culture

M14 cells were kindly provided by Dr. Robert Hoffman (University of California San Diego). As described in our previous studies, M14-OL, a M14 derivative cell line was established via three-rounds of in vivo passage, isolation and purification. The established OL cells, upon tail-vein injection, form limited number of metastatic foci (defined as oligometastatic–OL) on the mouse lungs [34]. Melanoma Stem Cells (OL-SCs) were isolated and purified from OL cells using the method we previously described [35, 69]. OL cells were maintained in DMEM high glucose supplemented (Gibico, USA) with 10% fetal bovine serum (FBS) (Gibico, USA) and 1% penicillin–streptomycin (Hyclone, USA). OL-SCs were cultured in DMEM/F12-based normal stem-cell media (Hyclone, USA) supplemented with 2% B27 (Gibico, USA) and 1% penicillin–streptomycin (Hyclone, USA).

Exosome isolation and identification

OL and OL-SCs cells were cultured in petri dishes (diameter:10 cm, Thermo Fisher Scientifc, USA) containing 12–15 ml of cell culture medium, and the culture medium of 10 dishes was collected each time for exosome extraction. Exosomes were obtained by differential ultracentrifugation. Cells were harvested when OL and OL-SCs cultures reached 90% confluency. After centrifugation at 800 g for 5 min, the supernatants were harvested and centrifuged at 2000 g at 4 °C for 10 min. The collected supernatant was rapidly concentrated using a centrifugal concentrator with a pore size of 100 kDa MWCO (Millipore, USA). For differential ultracentrifugation, the supernatants collected from low-speed centrifugation were centrifuged at 10000 g at 4 ̊C for 10 min and then passed through a 0.22 μm filter (EMD Millipore, USA). Thereafter, the supernatants were centrifuged at 100000 g at 4 ̊C for additional 70 min, the exosome pellets were washed with 2 ml of Phosphate-buffered saline (PBS, Hyclone, USA) and resuspended in 35 ml of PBS, and the sample was centrifuged again at 100,000 g for 70 min. The resultant pellets were resuspended in 200-300 μl PBS and stored at -80 ̊C for further analysis.

Protein content of the exosomes isolated were determined by using a micro-BCA Protein Assay Kit (CWBIO, China). Morphology of exosomes were analyzed using Transmission electron microscopy (TEM, JEM-1400PLUS, JEOL). The size distribution and concentration of exosomes were determined by Nanoparticle Tracking Analysis (NTA). Western blot(WB)was used to characterize the expression of exosomal markers.

Western blot(WB)

Total cell proteins were extracted by RIPA (Solarbio, China). Exosomal proteins were extracted by repeated freeze–thaw lysis at -80℃. Cellular and exosomal protein concentrations were measured by BCA protein assay kit (CWBIO, China) and micro-BCA protein assay kit, respectively. Each protein sample (20 μg) was separated by electrophoresis with 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Subsequently, PVDF membranes were blocked, incubated with antibodies and washed. Primary antibodies of Alix, CD63, CD81, Calnexin, RAB27a, and the loading controls (GAPDH and β-Actin) were purchased from Proteintech Group, USA. LC3, P62 and TSG101 antibody was purchased from Abcam, Britain. The secondary Antibodies: anti-Rabit and anti-Mouse was purchased from Proteintech Group.

Exosome treatment

Exosome treatment was used in migration and invasion Transwell assays and PCR experiments. Protein concentration of the prepared exosomes were determined by the micro-BCA Protein Assay Kit (CWBIO, China). For Transwell analysis, when treated with exosomes secreted by OL-SCs cells (OL-SCs-EXO), OL cells were seeded in the upper chamber of the chamber and co-cultured with OL-SCs-EXO (40 μg) for 24 h. When treated with exosomes derived from OL-SCs with knockdown of the RAB27a gene (OL-SCs-shRAB27a-EXO), the culture medium of the same number of shRAB27a-OL-SCs cells and shNC-OL-SCs cells was collected for the extraction of exosomes. During the Transwell migration and invasion assay, equal volumes of OL-SCs-shRAB27a-EXO, OL-SCs-shNC-EXO, and PBS were added to the upper chamber to co-culture with OL cells for 24 h. When OL cells were treated with exosomes secreted by OL-SCs cells transfected with NC-inhibitor and miR-1268a-inhibitor (OL-SCs-NC-inhibitor-EXO, OL-SCs-1268a-inhibitor-EXO). OL cells were seeded in the upper chamber of the chamber, and OL-SCs-NC-inhibitor-EXO and OL-SCs-1268a-inhibitor-EXO of the same quality (40 μg) were added to co-culture for 24 h. For PCR experiments, OL cells were seeded in six-well plates and cultured for 48 h, and OL-SCs-EXO (60 μg) and an equal volume of PBS were added to continue to culture for 24 h, and the cells were collected.

Transwell migration and invasion assays

Cell invasion and migration was performed in triplicate using 24-well Transwell plate (8 μm pore size, BD Falcon, USA), and were used to perform cell invasion assay with Matrigel (1:10 dilution in serum free medium, BD Biosciences, USA) and migration assay without Matrigel. For the migration assay, 30,000 cells were placed in the upper chamber supplemented with 300 μl serum-free DMEM medium and 800 μl DMEM medium containing 20% FBS in the lower chamber. After culturing for 24 h, the chamber was taken out and washed with PBS to remove floating cells. Subsequently, the cells on the chamber membrane were fixed with ice methanol for 20 min, washed twice with PBS to remove residual methanol, and stained with crystal violet for 5–10 min. Next, the stained chamber was washed twice with PBS to remove the floating color, and the cells on the membrane surface of the upper chamber were removed by gently wiping with a cotton swab. Finally, the dried chamber was photographed under a microscope and 10 random fields from three replicate wells were counted by the software image J. The basic steps of the invasion assay are similar to that of the migration assay. The difference is that in the invasion assay, the chamber needs to be coated with Matrigel 24 h in advance, and 50,000 cells were added. Each experiment was repeated 3 times.

Lentivirus and oligonucleotide transfection

The lentivirus particles of NC, miR-1268a overexpression (OE) and RAB27a silencing (sh-RAB27a) plasmids were purchased from Shanghai GenePhaema Company (China). miR-1268a mimics, miR-1268a inhibitor and negative control were chemically synthesized by GenePhaema (Shanghai, China). Cells were infected with lentivirus or transfected with siRNA and mimics by Lipofectamine 2000 (Thermo Fisher Scientifc, USA) according to the manufacturer's instructions, respectively. After 72 h of infection and 48 h of transfection, the overexpression and silencing of target genes or miRNAs were analyzed by PCR to verify the infection and transfection efficiency.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA from cells and exosomes was extracted using Trizol (Invitrogen, USA) and miRNeasy Serum/Plasma Kit (QIAGEN, Germany), respectively. Reverse transcription was performed using PrimeScript RT Master Mix (Takara, Japan) and Mir-X™ miRNA First Strand Synthesis Kit (Takara, Japan). RT-qPCR was conducted using SYBR Green Real-time PCR Master Mix Kit (Takara, Japan) according to the manufacturer’s instructions and the following PCR condition: in a 10 μl reaction volume, initial pre-incubation at 95 ℃ for 30 s, followed by 39 cycles at 95 ℃ for 5 s and 60 ℃ for 30 s.The relative expression level of miRNAs was calculated through normalization to U6 internal controls, and mRNAs were normalized with GAPDH. The primers used for PCR are shown in Table 1.

Table 1 PCR primer sequencemiRNA Sequencing and analysis

Triplicates of OL-SCs cell exosomes and OL cell exosomes extracted by ultracentrifugation were sent to BGI for miRNA sequencing and data analysis (https://biosys.bgi.com/). For miRNA sequencing, total RNA in OL and OL-SCs exosomes was extracted. Total RNA was identified and quantified using NanoDrop and an Agilent 2100 Bioanalyzer (Thermo Fisher Scientific, MA, USA). Library was prepared with 1 μg total RNA for each sample. Total RNA was purified by electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel and small RNA regions corresponding to the 18–30 nt bands in the marker lane (14–30 ssRNA Ladder Marker, TAKARA, Japan) were excised and recovered. Then the 18–30 nt small RNAs were ligated to adenylated 3’ adapters annealed to unique molecular identifiers (UMI), followed by the ligation of 5’adapters. The adapter-ligated small RNAs were subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen, USA), followed by several rounds of PCR amplification with PCR Primer Cocktail and PCR Mix to enrich for cDNA fragments. The PCR products were seperated by agarose gel electrophoresis. Target fragments (110 ~ 130 bp) were cut and purified by QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). Fragment size distribution was examined using an Agilent 2100 Bioanalyzer, and quantitative detection of the library was performed using quantitative real-time PCR (TaqMan probes). The final ligation PCR products were sequenced using the BGISEQ-500 platform (BGI-Shenzhen, China). For data analysis, the raw sequencing data is pre-processed by quality assessment and data filtering, and then the obtained sequences are aligned and annotated with the miRNA database to obtain known miRNAs. For the sequences that were not aligned and annotated in the sequencing results, they were compared with the whole genome sequence of the species, and the sequence was preliminarily judged to be an unknown new miRNA. Next, the samples (OL-SCs-EXO, n = 3; OL-EXO, n = 3) were used for comparative analysis of miRNA expression using the DEGseq toolkit. The filter condition is: |log2(FC)|> 2 (FC: fold-change), P < 0.001.

Detection of Cy5-labelled miR-1268a transfer

OL-SCs was transfected with Cy5 labeled miR-1268a (GenePhaema, China), and extracted its exosomes by the above method of separation and purification of exosomes. Then OL-SCs exosomes were stained with PKH26 (Sigma, USA) according to the manufacturer’s instructions. After OL was cultured on 24-well plate cell slides for 24 h and respectively co-cultured with stained exosomes (40 μg) for 6 h,9 h,12 h. Next, the cells were fixed with ice ethanol, washed with PBS, and the nucleus stained with DAPI. Images were acquired with a confocal fluorescence microscope.

Cell cycle

Cells were subjected to cell cycle analysis by flow cytometry. OL cells were transfected with miR-1268a-mimics and NC-mimics, respectively. 48 h after transfection, cells were harvested by centrifugation at 800 rpm/min for 5 min and residual medium was washed with PBS. Next, cells were fixed with 70% ethanol overnight at − 20 °C, washed with PBS, resuspended in 500 μl PBS, and then cells were stained with PI/RNase staining solution (1.25 mg/ml PI and 2 mg/ml RNase A) in the dark at 37 °C for 30 min. After incubation, cells were analyzed for DNA content using a flow cytometer (FACS Vantage SE, BD, America) and the results were analyzed using ModFit LT software. Triplicates were submitted for flow cytometry analysis.

CCK-8 Cell proliferation assay

Cell proliferation analysis was performed using the Cell Counting Kit-8(CCK-8, Beyotime, China). 2000 cells were seeded in each well of a 96-well plate, and five replicate wells were set in each group. After the cells adhered, 10 μl of CCK-8 reagent was added to each well and incubated at 37 °C for 2 h. The absorbance at 450 nm of each well was detected by a microplate reader. Cell proliferation activity was measured for 5 consecutive days and cell proliferation curve was plotted.

EdU analysis

Cell proliferation was detected using the BeyoClick™ EdU-488 Cell Proliferation Assay Kit (Beyotime, China). After OL cells were transfected with mimics for 24 h, pre-warmed EdU solution (10uM) was added and incubated for 2 h. Then the cells were collected and fixed with fixative (immunostaining fixative P0098) at room temperature for 15 min. The fixed cells were washed 3–5 times with washing solution (immunostaining washing solution P0106), treated with permeabilizing solution (immunostaining strong permeabilizing solution P0097) and incubated for 10–15 min. Then cells were washed once again. The Click reaction solution (Click Reaction Buffer, CuSO4, Azide 488, Click Additive Solution) was prepared according to the kit instructions. Finally, 0.5 ml of Click reaction solution was added to each well, and incubated at room temperature for 30 min in the dark. The Click reaction solution was removed by suction. After three washes, cells were observed under a fluorescence microscope and the fluorescence was detected by a multi-function microplate reader.

Animal experiments

All NOD-SCID mice used by experiments were housed in the Animal Experiment Center of Chongqing Medical University. Animal experiments were performed in accordance with approved protocols and in accordance with the Institutional Animal Welfare Guidelines of Chongqing Medical University, and all NOD-SCID mice used in the study were euthanized by cervical dislocation.\(5 \times 10^\) OL cells of stably transfected with the miR-1268a overexpression vector (OL-miR-1268a-OE) or the control lentiviral vector (OL-OE-NC), prepared in 100 μl PBS were transplanted into each male NOD/SCID mouse via tail intravenous injection, respectively (OE-1268a group: n = 6; OE-NC group: n = 6). Mice were weighted every 2 days and dissected when the weight loss lasted for one week. Lung-derived metastatic nodules were examined and quantified by whole-lung bright, fluorescent and H&E images.

Autophagy flux analysis of LC3B puncta

Autophagy flux analysis of LC3B puncta was conducted as we previously reported [34]. According to the manufacturer's instruction, the cell was infected with adenoviral-expressing mCherry-GFP-LC3B fusion protein (Beyotime, China) and the accumulation of autophagosomes is quantified by counting mRFP-LC3 puncta around the cells.

Transmission electron microscopy (TEM) analysis of autolysosome

Cells were pelleted by centrifugation at 1200 rpm/min for 10 min. Cells were fixed with 2.5% glutaraldehyde for 24 h and 1% osmium tetroxide for 1 h and rinsed with 0.1 M phosphoric acid rinse. Subsequently, cells were dehydrated sequentially with graded ethanol (50%, 70%, 90%) and 90% acetone at 4 °C, and 3 times with 100% acetone at room temperature. Next, the cells are embedded, solidified and sectioned. Sections were stained with 3% uranyl acetate-lead citrate and photographed with a JEM-1400Plus transmission electron microscope (JEOL Ltd, Japan) to observe autolysosome.

Statistical analysis

All statistical analysis in this study were performed with GraphPad Prism 8.0 software and presented as mean ± SD. Data were analyzed for normal distribution by Normality and Log Normality tests or Normality of residuals (Q-Q Plots, Anderson-Daring, D’Agostino & Pearson test, Shapiro–Wilk test, Kolmogorov–Smirnov test). Two groups were compared using Student's t test, and multiple groups were analyzed using one-way analysis of variance (ANOVA). All experiments were repeated at least three times. P < 0.05, P < 0.01 and P < 0.001 were considered statistically significant and were marked with an asterisk, double asterisks and triple asterisks, respectively.