Research object
In this study, 34 tumor patients treated in Nanjing Drum Tower Hospital from May 2021 to September 2021 were selected. The average age of tumor group was 59 years old, with 25 male and 9 female. There were 13 cancer types in cancer group, including gastric cancer, lung cancer, colorectal cancer and so on. The average age of healthy group was 49 years old, with 18 male and 16 female. Details of the study subjects are provided in Supplementary Tables 1–2. This study was approved by the Ethics Committee of Nanjing Drum Tower Hospital. All methods were performed in accordance with the relevant guidelines and regulations.
Cell Culture
The cell lines A549, Beas-2b, SGC7901 and GES cells were purchased from Cobioer bioscience (Nanjing, China). A549 and Beas-2b cells were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS), while SGC7901 and GES were cultured in RPMI-1640 medium (Gibco, USA) with 10% FBS. All cells were maintained in a humidified incubator (Thermofisher, SHKE8000-8CE) at 37℃ with 5% CO2.
Exosomes Isolation From Cells
The cells were cultured with correspond media contain 10% exosome-free FBS medium for 24 h, when it’s grown in common media for 50–60%. Then the medium was collected and centrifuged at 300 g for 10 min, followed by 2000 g for 30 min to remove cell debris. After that the supernatant was filtered using a 0.22 µm pore filter (Merck Millipore, German) to remove the apoptotic bodies, shedded vesicles and cell debris. The collected filtered supernatant was centrifuged at 100,000 g for 70 min at 4°C (Beckman Coulter Optima XE-100, Type 90 Ti rotor) twice to collect EVs. The pelleted EVs were resuspended in 100 µL phosphate-buffered saline (PBS).
Exosomes Isolation From Human Serum Samples
The collected serum was centrifuged at 300 g for 10 min and 2000 g for 30 min at 4°C to remove the cell and cell debris before storing at -80°C. For the isolation of exosomes from serum, the frozen serum was thawed at 4°C and centrifuged at 10,000 g for 10 min at 4°C. After that, the supernatant was filtered through a 0.22 µm pore filter to remove residual contamination. Then, the supernatant was centrifuged at 100,000 g for 70 min at 4°C twice to collect EVs, the pellets resuspended in 100 µL phosphate-buffered saline (PBS).
Nanoparticle Tracking Analysis (Nta)
The concentration and size distribution of exosomes were measured using Zetaview (German, PMX120). The samples were diluted 1000-fold with PBS for detection. Each sample was diluted in triplicate and each diluted sample was analyzed 3 times using the same settings for detection.
Transmission Electron Microscopy
The cell-derived exosomes (5⋅109/mL) were used for transmission electron microscopy (TEM) to examine the morphology and size of exosomes. Exosomes sample was deposited onto the ultrathin copper mesh. The TEM experiments were performed at the Analysis Center of Southeast University.
Quantitative Real-time Pcr (Qrt-pcr)
Total cellular RNA was extracted from cells with TRIzol reagent (Invitrogen, USA) and then subjected to reverse transcription using a PrimeScript™ RT Master Mix Kit
(Takara, Japan). qRT-PCR was performed on a Step One Plus™ PCR system (Applied Biosystems, 7500) using ChamQ™ SYBR® qPCR Master Mix (Vazyme, China), follow the instructions of the guidance. The primer sequences were as follows: CEA, forward 5′- GCACCTCAGACCAATCATCAACT-3′, reverse 5′- CCACTTCTCAAGGACCAAATACAC-3′; GAPDH, forward 5′-GAAGGTGAAGGTCGGAGTCA-3′, reverse 5′-TTGAGGTC AATGAAGGGGTC-3′
Western Blot
Western Blot
Cells were lysed in RIPA buffer containing 1mM PMSF and the protein concentration was quantified using the BCA protein assay reagent kit. The samples were separated by SDS-PAGE and transferred to PVDF membrane. Then, the membrane was blocked with 5% non-fat milk and subsequently incubated with primary antibody as follows: CD63 (ab134045, Abcam), TSG101 (ab125011, Abcam), Calnexin (ab133615, Abcam) and GAPDH (10494-1-AP, Proteintech). Afterwards, the membrane was incubated with the HRP conjugated secondary antibody at room temperature for 1 h. The bands were detected by the ultra-ECL regent kit (Beyotime Shanghai, China).
Nucleic Acid To Protein Ratio (Npr) Measurement
Since nucleic acid and protein have the maximum absorption peaks at 260 and 280 nm, respectively, the maximum 260 nm-280 nm absorbance ratio of exosomes was analyzed to assess the nucleic acid and protein ratio of exosomes which called NPr. To detect the NPr of cell and serum-derived exosomes, 1.5 µL exosomes were directly aspirated to determine the ratio of absorbance at 260 nm to 280 nm by a UV absorption spectrophotometer (Nanodrop 2000, USA). For the detection of CEA+ exosomes, CEA+ exosomes were isolated from serum using magnetic bead modify with CEA antibody (Siemens, USA) to detect their NPr. Briefly, 100 µL of serum was mixed with 250 µL of magnetic bead modify with CEA antibody in a 1.5 mL tube and incubated on a rotator for 30 minutes at room temperature. The tubes were placed in a magnetic field and the supernatants were removed after standing for 1 minute. Then, the magnetic beads were washed 3 times with 200 µL of PBST, dissolved in 20 µL of DEPC water, and heated at 95°C for 10 minutes to dissociate nucleic acids and proteins from the magnetic bead. The supernatant was separate by magnetic rack. Finally, as described above, 1.5 µL of supernatant was taken to detect the NPr of CEA+ exosomes.
Double-stranded Dna To Protein Ratio (Dsdpr) Measurement
The isolation of CEA+ exosomes followed the NPr assay procedure. The absolute quantitative of dsDNA and protein were carried out according to the product instructions (Thermofisher, USA). Specifically, 1 µL of CEA+ exosomes lysis was combined with 199 µL of working solution, then vortexed and incubated for 2 min (dsDNA) or 15 min (proteins) at room temperature. The Qubit Flex Fluorometer was used to quantify the sample concentration.
Statistical Analysis
All calculations were performed by GraphPad Prism 9. Statistical analyses were performed using Student’s t-test, ANOVA and Fisher’s extract test. P < 0.05 was considered statistically significant. The results are expressed as the mean ± SEM for three independent experiments.