Ev depleted fbs

For EV isolation, A549 and H1299 adherent BCCs were seeded in 150 mm dishes with growth media supplemented with 10% EV-depleted FBS (System Biosciences) and CSCs were seeded in T75 low adherence flask with EV-free CSC media (serum-free DMEM-F12 medium; Gibco-Invitrogen) containing 0.4% Albumin Bovine Fraction V (Sigma-Aldrich), B-27 Supplement (Gibco-Invitrogen), 20 μg/mL EGF (PeproTech), and 10 μg/mL bFGF (PeproTech). The media was collected after 48 hours and centrifuged at 300 × g for 10 minutes at 4°C. The supernatant was transferred to a new polypropylene tube and centrifuged at 2,000 × g for 20 minutes at 4°C. The supernatant then was filtered through a 0.22 μm polyethersulfone membrane filter (Corning Incorporated) and transferred to a polyallomer ultracentrifuge tube and centrifuged at 10,000 × g for 30 minutes at 4°C (Beckman Coultier Optima Max Ultra). The supernatant was then transferred to a new polyallomer ultracentrifuge tube and centrifuged at 100,000 × g for 70 minutes at 4°C (Beckman Coultier Optima Max Ultra). The pellet was then suspended in 1X PBS, and the ultracentrifugation step was repeated to obtain an EV pellet that was suspended in 200 μL of sucrose buffer (5% sucrose, 50 mmol/L Tris, and 2 mmol/L MgCl).

Plasma exosome isolation was performed using the ExoQuick exosome precipitation kit (SBI System Biosciences, Mountain View, CA, USA) according to the manufacturer’s protocol [26 (link), 27 (link)]. HepG2 cell lines constantly expressing CrebH were seeded in a 10 cm culture dish overnight and changed with fresh DMEM containing 10% EV-depleted FBS (SBI System Biosciences) for 48 h. The culture medium was centrifuged at 1000 × g for 5 min at 4 ℃ to eliminate suspended cells, filtered through a 0.22 μm syringe filter, and transferred to a Macrosep 100 KD filter system (PALL Laboratory, MA, USA) to enrich the particles with 30–90 nm molecular size. The exosomes contained in the enriched particles were isolated using ExoQuick-TC exosome precipitation solution (SBI System Biosciences). The exosome pellet was resuspended in 100 μL filtered-PBS and stored at − 80 ℃.

Hyaluronic acid (HA, Mw = 4.8 kDa), 3-(diethylamino)propylamine (DEAP), N-hydroxysuccinimide (NHS), N,N’-dicyclohexylcarbodiimide (DCC), triethylamine (TEA), deoxycholic acid (DOCA), 4-dimethylaminopyridine (DMAP), pyridine, dimethyl sulfoxide (DMSO), sodium tetraborate, adipic acid dihydrazide (ADH), doxorubicin hydrochloride (DOX), paraformaldehyde, heparin, and Triton X-100, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorin e6 (Ce6) was purchased from Frontier Scientific Inc (Logan, UT, USA). RPMI-1640 medium, DMEM medium, fetal bovine serum (FBS), phosphate buffered saline (PBS), ethylene diamine tetra-acetic acid (EDTA), penicillin, trypsin, and streptomycin were purchased from Welgene Inc (Seoul, Korea). EV-depleted FBS was purchased from System Biosciences Inc. (Palo Alto, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc. (Santa Clara, CA, USA). Wheat Germ Agglutinin Alexa Fluor^® 488 Conjugate (WGA-Alexa Fluor^® 488), fluorescein isothiocyanate (FITC) were purchased from Life Technologies (Carlsbad, CA, USA).