6 well ultra low adherent plate

Tumor self-renewing and anchorage-independent spheroids were obtained by culturing breast cancer cells MCF7, Hs587T and MDA231; melanoma cells SBcl2 and FM6; human breast cancer stem cells and pancreatic cancer stem cells in stem cell-selective conditions according to the manufacturer’s instructions (StemCell Technologies, Tukwila, WA). Briefly, cancer and cancer stem cells were propagated in 6-well ultra-low adherent plates (Corning) in Complete MammoCult Medium (Human) by adding 50 mL of MammoCult Proliferation Supplements to 450 mL of MammoCult Basal Medium (StemCell Technologies). The following were added to obtain Complete MammoCult Medium: 4 ug/mL Heparin (StemCell Technologies), 0.48 μg/mL hydrocortisone (StemCell Technologies), 200 U penicillin/ml; and 200 μg streptomycin/ml (Invitrogen).

EWS cells (2 × 10⁴ cells/well) were seeded in a 6-well ultra-low adherent plate (Corning) and pretreated with exosomes (0 or 20 μg/mL) in a sphere assay. Following 5 days, cells were harvested and lysed for use in western blot. Total protein was isolated from cells and exosomes using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Protein from cells was quantified using the BCA method (Pierce, Waltham, MA, USA) and the amount of protein in exosomes was estimated by measuring protein content with the Bradford assay (Bio-Rad, Hercules, CA, USA). SDS-PAGE was conducted under reducing or non-reducing conditions followed by immunoblot as previously described [43 (link)]. Antibodies used in this experiment were TSG101, CD63, Calnexin (1:500, all Santa Cruz Biotechnology, Dallas, TX, USA), HIF-1α (1:1000, BD Biosciences, San Jose, CA, USA), CASP8AP2/FLASH (1:1000, Abcam, Cambrige, UK) and α-Tubulin (1:4000, Sigma Aldrich, St. Louis, MO, USA). Blots were incubated with appropriate horseradish peroxidase-tagged secondary antibodies and visualized using the _MYECL Imager (Thermo Fisher Scientific, Waltham, MA, USA).