Total exosome isolation

Peripheral blood samples from AMI patients were collected at admission and after PCI (at the time of discharge); while samples from HCs were collected at the time of enrollment. After sample collection, the serum was immediately isolated. Then, the exosomes were separated from the serum through Total Exosome Isolation (from serum) (No. Cat. 4478360, Invitrogen™, Waltham, USA). All test procedures were performed in strict accordance with the instructions.
Serum exosomal miR-186-5p was detected using a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. The U6 was used as the internal reference, and the final results were calculated using the 2^−ΔΔCt method. The primer sequences of serum exosomal miR-186-5p and U6 were the same as in our previous study (17 (link)).

Exosomes were isolated from conditioned media of PT-sen and PT-res ITGA6WT and KO cells following manufacturer’s indications for Total Exosome Isolation (from cell culture media) Kit (Invitrogen, 4478359). Briefly, conditioned media were centrifuged to remove cells and debris, then the supernatant was transferred to a new tube. Half volumes of Total Exosome Isolation reagent was added to the cell-free culture media: the mixture was thoroughly mixed and incubated overnight at 4 °C. The day after, the mixture was centrifuged to precipitate exosomes that will be contained in the pellet. The exosomes resuspended in 1X PBS were then used for characterization and functional assays. To evaluate ITGA6 levels, exosomes were lysed using cold RIPA buffer with protease inhibitors, protein concentration was determined by Bio-Rad protein assay (Bio-Rad) and then protein levels were evaluated by western blot analysis. ITGA6 proteins levels in exosomes upon CDDP treatment was evaluated adding the drug directly to serum-free media for the indicated time points before conditioned media harvesting. To evaluate the functional role of ITGA6 contained in exosomes, the resuspension was added to serum-free media to challenge TOV-112D PT-sen cells for 16 h which then were used to evaluate ovaryspheres formation ability on mesothelial cells, as described above.

The HepG2 cell line was purchased from the ATCC. Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (Gibco) was used to culture HepG2 cells. The following EVs isolation kits were used: ExoQuick-TC exosome precipitation solution (EXOTC50A-1, System Biosciences, Palo Alto, CA, United States), Total Exosome Isolation (from cell culture medium; 4478359, Invitrogen, Carlsbad, CA, United States), and exoEasy Maxi Kit (76064, EM, Qiagen, Hilden, Germany). After EVs isolation, EVs proteins were extracted with RIPA lysis buffer (Sangon Biotech, Shanghai, China) supplemented with protease inhibitor cocktail (Selleck Chemicals, Houston, TX, United States) for western blot analysis using rabbit polyclonal anti-CD63 (A5271, ABclonal Technology, Woburn, MA, United States) and rabbit anti-tumor susceptibility 101 (TSG101) (HPA006161, Sigma-Aldrich, St. Louis, MO, United States) antibodies.

Peripheral blood samples (6 ml) were collected in EDTA tubes. After centrifugation at 3,000 × g at 4°C for 15 min, at least 3 ml plasma was collected and stored at −80°C. After thawing at 37°C, the plasma was centrifuged at 2,000 × g for 20 min, and the supernatant was obtained. The debris was centrifuged again at 10,000 × g for 20 min. Then, Total Exosome Isolation (from plasma) reagent (Invitrogen, 4484450, California, United States) (Lobb, et al., 2015 (link)) was used according to the manufacturer’s instructions. Briefly, protease K (0.05 times the volume of plasma) was added to the plasma and incubated at 37°C for 10 min. Then, 0.2 volumes of exosome extraction reagent were added to the mixture and incubated at 4°C for 30 min. The mixture was centrifuged at 10,000 × g at room temperature for 5 min. The exosomes, contained in a pellet at the bottom of the tube, were resuspended in PBS and stored at −80°C.

Exosomes from all samples of malignant ascites or peritoneal washings were prepared using Total Exosome Isolation (from body fluids) (Catalog Number: 4484453, Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions, with some modifications (23 (link)). Briefly, all samples in sterilized 50-mL centrifuge tubes were centrifuged at 2,000 × g for 30 min to remove floating cells and debris. Then, supernatants were thoroughly mixed with 0.5 volumes of Total Exosome Isolation Reagent (from other body fluids) by pipetting repeatedly until the solution was homogenous, and samples were incubated at room temperature for 30 min. After incubation, the mixed solutions were centrifuged at 10,000 × g at room temperature for 10 min (XE-90; Beckman Coulter, Brea, CA, USA). The exosome pellets were washed and re-suspended in 100 µL of phosphate-buffered saline (PBS) for quantification using a CD63 exoELISA (catalog# EXOEL-CD63A-1; System Biosciences, Mountain View, CA, USA) according to the manufacturer’s instructions (24 (link)). Finally, the purified exosomes were re-suspended in PBS at a concentration of ~10¹⁰ exosome particles per milliliter and stored at −80°C until use.

EV were isolated from 500 to 1500 µL of pre-surgery serum samples of all the cases of colon carcinoma patients having mutation in the tissue DNA using Invitrogen Total Exosome Isolation (Vilnius, Lithuania) according to the manufacturer’s instructions. Hemolyzed serum samples were excluded. Size distribution analysis of EV extracted from two random samples was performed using dynamic light scattering with a ZetaSizer (Malvern Instruments, Worcestershire, UK). Samples were diluted in phosphate-buffered saline (PBS) and 3 × measurement runs were performed under standard settings (refractive index: 1.331, viscosity: 0.89, temperature: 25 °C).